Inducible, Tunable, and Multiplex Human Gene Regulation Using CRISPR-Cpf1

ABSTRACT

Drug-inducible, tunable, and multiplexable Clustered Regularly Interspaced Short Palindromic Repeats from  Prevotella  and  Francisella  1 (Cpf1)-based activators, and methods of use thereof.

CLAIM OF PRIORITY

This application is divisional application of U.S. patent application Ser. No. 16/606,680, which is a national stage application under 35 USC § 371 of International Application No. PCT/US2018/028898, filed Apr. 23, 2018, which claims the benefit of U.S. Patent Application Ser. No. 62/488,585, filed on Apr. 21, 2017.

The entire contents of the foregoing are hereby incorporated by reference.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under Grant Nos. GM107427 and GM118158 awarded by the National Institutes of Health. The Government has certain rights in the invention.

SEQUENCE LISTING

This application contains a Sequence Listing that has been submitted electronically as an XML file named “40978-0278002_SL_ST26.XML.” The XML file, created on Jun. 22, 2023, is 114,826 bytes in size. The material in the XML file is hereby incorporated by reference in its entirety.

TECHNICAL FIELD

Described herein are drug-inducible, tunable, and multiplexable Cpf1-based activators, and methods of use thereof.

BACKGROUND

RNA-guided CRISPR nucleases have revolutionized both biology and therapeutics with their ease of reprogrammability to recognize target DNA sequences. The widely used Cas9 from Streptococcus pyogenes (SpCas9) can be targeted to a specific DNA sequence with an associated complementary guide RNA (gRNA) provided that a protospacer adjacent motif (PAM) of the form NGG is also present.

SUMMARY

The present invention is based, at least in part, on the development of constitutively active and chemically inducible dCpf1-based transcriptional activator platforms, and methods of use thereof, including methods that use multiplex Cpf1 gRNA expression to achieve synergistic or combinatorial activation of endogenous genes in human cells.

Thus, provided herein are fusion proteins that include a catalytically inactive (i.e., catalytically inactive for DNA endonuclease activity) Clustered Regularly Interspaced Short Palindromic Repeats from Prevotella and Francisella 1 (Cpf1) from Lachnospiraceae bacterium ND2006 protein Cpf1 (dLbCpf1) fused to at least one activation domain (e.g., 1, 2, 3, 4, or more activation domains), preferably wherein the activation domain is a synthetic VPR activator (i.e., comprising four copies of VP16, a human NF-KB p65 activation domain, and an Epstein-Barr virus R transactivator (Rta)). Other activation domains include VP64, Rta, NF-KB p65, and p300, with optional intervening linkers between the Cpf1 and/or each activation domain.

Further, provided herein are fusion proteins that include a catalytically inactive Lachnospiraceae bacterium ND2006 Cpf1 (dLbCpf1) fused to a conditional dimerization domain, with optional intervening linkers between the Cpf1 and/or each activation domain. In some embodiments, the conditional dimerization domain is DmrA or DmrC. These fusion proteins can be provided in compositions or kits that also include a second fusion protein comprising at least one activation domain (e.g., 1, 2, 3, 4, or more activation domains) fused to a second conditional dimerization domain that dimerizes with the conditional dimerization in the fusion protein of claim 2 in the presence of a dimerizing agent, with an optional intervening linker between each of the activation domain(s) and/or the second dimerizing domain. In some embodiments, the conditional dimerization in the fusion protein of claim 2 is DmrA, and the second conditional dimerization domain is DmrC, or (ii) the conditional dimerization in the fusion protein of claim 2 is DmrC, and the second conditional dimerization domain is DmrA. In some embodiments, the activation domain is VP64, Rta, NF-κB p65, VPR, or p300.

Also provided are kits comprising the fusion proteins, and/or nucleic acids encoding a fusion protein as described herein, optionally with the dimerizing agent.

Also provided herein are nucleic acids encoding the fusion proteins described herein, vectors comprising the nucleic acids, and cells comprising the nucleic acids and/or vectors and optionally expressing the fusion proteins.

Further, provided are methods for increasing expression of a target gene in a cell, the method comprising contacting the cell with, or expressing in the cell, one or more of:

-   -   (i) a fusion protein that includes a catalytically inactive         Lachnospiraceae bacterium ND2006 protein Cpf1 (dLbCpf1) fused to         at least one activation domain (e.g., 1, 2, 3, 4, or more         activation domains), and at least one crRNA that directs the         fusion protein to a regulatory region, e.g., a promoter region,         of the target gene; and/or     -   (ii) a first fusion protein that includes a catalytically         inactive Lachnospiraceae bacterium ND2006 Cpf1 (dLbCpf1) fused         to a conditional dimerization domain, with optional intervening         linkers between the Cpf1 and/or each activation domain, and a         second fusion protein comprising an activation domain fused to a         second conditional dimerization domain that dimerizes with the         conditional dimerization in the first fusion protein in the         presence of a dimerizing agent (with an optional intervening         linker between the activation domain and the second dimerizing         domain), and at least one crRNA that directs the fusion protein         to a regulatory region, e.g., a promoter region, of the target         gene.

Also provided are methods of increasing expression of a plurality of target genes (e.g., two, three, four, or more) in a cell, the method comprising contacting the cell with, or expressing in the cell, one or more of:

-   -   (i) a fusion protein that includes a catalytically inactive         Lachnospiraceae bacterium ND2006 protein Cpf1 (dLbCpf1) fused to         at least one activation domain (e.g., 1, 2, 3, 4, or more         activation domains), and at least one nucleic acid encoding a         plurality of crRNAs that each direct the fusion protein to a         regulatory region, e.g., a promoter region, of one of the target         genes;     -   (ii) a first fusion protein that includes a catalytically         inactive Lachnospiraceae bacterium ND2006 Cpf1 (dLbCpf1) fused         to a conditional dimerization domain, with optional intervening         linkers between the Cpf1 and/or each activation domain, and a         second fusion protein comprising an activation domain fused to a         second conditional dimerization domain that dimerizes with the         conditional dimerization in the first fusion protein in the         presence of a dimerizing agent, and at least one nucleic acid         encoding a plurality of crRNAs that each direct the fusion         protein to a regulatory region, e.g., a promoter region, of one         of the target genes.

In some embodiments, the cell is a mammalian cell, e.g., a human cell.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

DESCRIPTION OF DRAWINGS

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

FIGS. 1A-1E. Targeted human endogenous gene regulation using individual crRNAs with dLbCpf1-based activators

-   -   (A) Schematic showing direct dLbCpf1-VPR and dLbCpf-p65         activator fusion proteins.     -   (B) Activities of dLbCpf1-p65 or dLbCpf1-VPR using single crRNAs         at three endogenous human genes (HBB, AR, and NPY1R) in human         HEK293 cells. Relative activation of the indicated gene         promoters was measured by RT-qPCR. Three separate individual         crRNAs were targeted within promoter sequence 1 kb upstream of         the transcription start site for each gene. Relative mRNA         expression is calculated by comparison to the control sample in         which no crRNA is expressed. (C) Analysis of various crRNAs         targeted to the human CD5 and CD22 promoters. 32 crRNAs (16 for         each gene) were designed to target sites located within promoter         sequences 1 kb upstream or 500 bps downstream of the TSS of each         gene. Repetitive sequences were not targeted. After 72 hours         post-transfection, cells were stained with fluorescently labeled         antibody for CD5 or CD22 protein and fluorescent-positive cells         were quantified by flow cytometry. Error bars represent s.e.m.         for three biological replicates. Black circles indicate samples         that are significantly different (Student t-test, two-tailed         test assuming equal variance, p<0.05) compared to controls in         which no crRNA was expressed. (D) Schematic illustrating         drug-inducible bi-partite dLbCpf1-based activator fusion         proteins. dLbCpf1 is fused to one to four DmrA domains and VPR         or p65 is fused to a DmrC domain. Because DmrA and DmrC interact         only in the presence of an A/C-heterodimerizer drug (red         diamond), the bi-partite activator is only reconstituted in the         presence of the drug. (E) Activities of drug-inducible         bi-partite dLbCpf1-based activators using single crRNAs at three         endogenous human genes (HBB, AR, and NPY1R) in human HEK293         cells. Relative activation of the indicated gene promoters was         measured by RT-qPCR. The crRNA used for each promoters was the         one that showed the highest activity in (B) above. Data shown         in (B) and (E) represent three biological independent replicates         and error bars indicate standard deviation (SD) of three         technical replicates.

FIGS. 2A-2I. Multiplex and synergistic regulation of endogenous human genes by dLbCpf1-based activators

(A) Schematic of an expression cassette designed to express multiple gRNAs encoded on a single transcript. The arrows indicate cleavage sites being processed by the RNase activity of dLbCpf1. (B) Schematic illustrating multiplex expression of three crRNAs each targeted to a different endogenous gene promoter in a single cell. (C) Simultaneous activation of three endogenous human genes using crRNAs expressed from a multiplex transcript or from individual transcripts with dLbCpf1-VPR direct fusions (left panel), dLbCpf1-DmrA(×4) and DmrC-VPR fusions (middle panel), and dLbCpf1-DmrA(×4) and DmrC-p65 fusions (right panel). Transcripts were measured in HEK293 cells using RT-qPCR with relative mRNA expression calculated by comparison to the control sample in which no crRNA is expressed. (D) Activities of MST crRNA with different dLbCpf1-based activators in human U20S cells. Graphs showing activation of three endogenous human genes with dLbCpf1-VPR direct fusions, dLbCpf1-DmrA(×4) and DmrC-p65 fusions, and dLbCpf1-DmrA(×4) and DmrC-VPR fusions and crRNAs expressed from a multiplex single transcript (MST) or from transcripts encoding a single crRNA. RNA expression was measured by RT-qPCR and relative expression shown was calculated by comparison to a control sample in which no crRNA is expressed. (E) Schematic illustrating multiplex expression of three crRNAs each targeted to the same endogenous gene promoter in the same cell. (F) Activities of direct dLbCpf1-p65 or dLbCpf1-VPR fusions with sets of three crRNAs expressed from a multiplex transcript or from individual transcripts on the HBB, AR, or NPY1R endogenous gene promoters. Transcripts were measured in HEK293 cells using RT-qPCR with relative mRNA expression calculated by comparison to the control sample in which no crRNA is expressed. (G) Activities of dLbCpf1-DmrA(×4) and DmrC-VPR fusions or with dLbCpf1-DmrA(×4) and DmrC-p65 fusions with sets of three crRNAs expressed from a multiplex transcript or from individual transcripts on the HBB, AR, or NPY1R endogenous gene promoters. Transcripts were measured in HEK293 cells using RT-qPCR with relative mRNA expression calculated by comparison to the control sample in which no crRNA is expressed. Data shown in (C), (D), (F) and (G) represent three biological independent replicates and error bars indicate standard deviation (SD) of three technical replicates. hU6, human U6 Polymerase III promoter; DR; direct repeat sequence. (H) Inducibility and reversibility of A/C heterodimerizer drug-regulated dLbCpf1-based activators. To measure the kinetics of activator induction, HEK293 cells were transfected with plasmids expressing dLbCpf1-DmrA(×4), DmrC-p65, and MST crRNAs targeting the human HBB or AR promoters. 34 hours after transfection, these cells were split into two cultures: one with media containing A/C heterodimerizer (500 uM) (top, black) and one with media lacking the A/C heterodimerizer (bottom, grey). Cells were collected at various time points and relative mRNA expression levels were measured by RT-qPCR compared to a negative control. (I) To measure the kinetics of reversibility, HEK293 cells were transfected as in (H). 24 hours after transfection, A/C heterodimerzer (500 uM) was added to the medium. 10 hours later, these cells were split into two cultures: one with media containing A/C heterodimerizer (500 uM) (top, blue) and one with media lacking the A/C heterodimerizer (bottom, purple). Cells were collected at various time points and relative mRNA expression levels were measured by RT-qPCR compared to a negative control. Error bars represent s.e.m. of three biological replicates.

DETAILED DESCRIPTION

Catalytically inactive forms of SpCas9 (“dead” SpCas9 or dSpCas9) nucleases have been fused with transcriptional activator or repressor domains to alter the expression of genes individually or genome-wide for library screens in mammalian cells¹⁻⁴, although efficient activation has required multiple regulatory domains to be recruited to a single promoter⁵. Both small molecule- and light-inducible dCas9-based gene regulatory fusions have also been described, providing additional important capabilities to this platform⁶⁻¹⁰. Recently described CRISPR-Cpf1 nucleases offer important additional capabilities beyond those of SpCas9 including shorter length gRNAs, the capability to target alternative T-rich PAM sequences, and processing of multiple guide RNAs from a single transcript by the Cpf1 nuclease itself¹¹. However, to the present inventors' knowledge “dead” Cpf1-based gene regulators have only been used to date to repress gene expression in bacteria¹² and a plant (Arabidopsis)¹³ and have not been shown to work in mammalian cells or for activating a target gene. Herein we describe constitutively active and chemically inducible dCpf1-based transcriptional activator platforms and show that multiplex Cpf1 gRNA expression can be leveraged to achieve synergistic or combinatorial activation of endogenous genes in human cells.

To our knowledge, the results reported here provide the first demonstrations that RNA-guided Cpf1-based fusions can be used to activate endogenous gene expression in any cell type and the first use of Cpf1-derived gene regulatory proteins in human cells. The orthogonal PAM recognition specificities of LbCpf1 compared with SpCas9 (TTTN versus NGG) open up a new range of targetable sequences for RNA-guided activator proteins. Given the reported higher genome-wide specificities of LbCpf1 compared with SpCas9^(14,15) dLbCpf1 activators may have comparable specificities to dSpCas9 activators, which have been shown by RNA-seq to cause few, if any, off-target gene activation events.

The present work also established drug-inducible dLbCpf1 activators that can be used to control gene regulation in multiple ways. These activators include paired fusion proteins, a first fusion protein that includes a catalytically inactive Lachnospiraceae bacterium ND2006 Cpf1 (dLbCpf1) fused to a conditional dimerization domain, with optional intervening linkers between the Cpf1 and/or each activation domain, and a second fusion protein comprising an activation domain fused to a second conditional dimerization domain that dimerizes with the conditional dimerization in the first fusion protein in the presence of a dimerizing agent, with an optional intervening linker between the activation domain and the second dimerizing domain. The bi-partite nature of these activators enables turning their activity on or off with a cell-permeable A/C dimerizer drug, which provides a useful capability for experimental systems. In addition, the level of activation desired can be tuned by increasing the number of DmrA dimerizer domains fused to dLbCpf1, which presumably leads to recruitment of increasing numbers of DmrC-activator fusions to a given promoter. Changing the activation domain used in the DmrC fusion influenced the extent of activation observed. Somewhat surprisingly and in contrast to previous results with dSpCas9-based activators, the p65 domain (with individual crRNAs) more consistently activated the three gene promoters examined than did the synthetic VPR activator (which contains six strong activation domains). With multiple crRNAs directed to a target promoter, the p65 domain provided stronger activation for the two genes on which synergism was observed. The ability to use the naturally occurring p65 activation domain rather than the synthetic VPR is advantageous as it avoids undesirable side effects (e.g., squelching) caused by very potent activators^(16,17) Beyond its utility for gene activation, this drug-inducible, multiplex dCpf1-based platform can be used to enable targeted recruitment of other heterologous proteins or functional domains to any endogenous genomic locus of interest.

The present work also demonstrated that a key advantage of the Cpf1 platform, the ability to more simply encode multiple crRNAs on a single transcript, can be leveraged to achieve multiplex activation of endogenous human genes in the same single cell. Multiplex regulation using dSpCas9 gene regulatory proteins is challenging due to substantial recombination between promoters if gRNAs are expressed from separate promoters¹⁸ or the need for additional accessory RNA sequences, promoters, or trans-acting factors if multiple gRNAs are expressed from a single transcript¹⁹⁻²⁵.By contrast, the shorter ˜40 nt length of crRNAs used by Cpf1 enables two or three crRNAs to be readily encoded on a single oligonucleotide thereby enabling the leveraging of chip-based synthesis to construct precise user-specified combinations of crRNAs targeted to genes of interest; doing the same with dSpCas9 gRNAs is more challenging due to the longer-length (˜100 nt) guide RNAs required and the accessory sequences and factors required to enable processing from a single transcript. Thus, these results demonstrate the feasibility of performing methods including multiplex library screens in which the expression of two or more genes are simultaneously regulated, thereby enabling the analysis of more complex cellular phenotypes using this approach.

Cpf1

Clustered, regularly interspaced, short palindromic repeat (CRISPR) systems encode RNA-guided endonucleases that are essential for bacterial adaptive immunity²⁶. CRISPR-associated (Cas) nucleases can be readily programmed to cleave target DNA sequences for genome editing in various organisms²⁷⁻³⁰. One class of these nucleases, referred to as Cas9 proteins, complex with two short RNAs: a crRNA and a trans-activating crRNA (tracrRNA)^(31,32). The most commonly used Cas9 ortholog, SpCas9, uses a crRNA that has 20 nucleotides (nt) at its 5′ end that are complementary to the “protospacer” region of the target DNA site. Efficient cleavage also requires that SpCas9 recognizes a protospacer adjacent motif (PAM). The crRNA and tracrRNA are usually combined into a single ˜100-nt guide RNA (gRNA)^(31,33-35) that directs the DNA cleavage activity of SpCas9. The genome-wide specificities of SpCas9 nucleases paired with different gRNAs have been characterized using many different approaches³⁶⁻³⁹. SpCas9 variants with substantially improved genome-wide specificities have also been engineered⁴⁰⁻⁴¹.

Recently, a Cas protein named Cpf1 has been identified that can also be programmed to cleave target DNA sequences⁴²⁻⁴⁵. Unlike SpCas9, Cpf1 requires only a single 42-nt crRNA, which has 23 nt at its 3′ end that are complementary to the protospacer of the target DNA sequence⁴⁴. Furthermore, whereas SpCas9 recognizes an NGG PAM sequence that is 3′ of the protospacer, AsCpf1 and LbCp1 recognize TTTN PAMs that are found 5′ of the protospacer⁴⁴. Early experiments with AsCpf1 and LbCpf1 showed that these nucleases can be programmed to edit target sites in human cells⁴⁴ but they were tested on only a small number of sites. On-target activities and genome-wide specificities of both AsCpf1 and LbCpf1 were characterized in Kleinstiver & Tsai et al., Nature Biotechnology 2016.

Provided herein are fusion proteins comprising LbCpf1. The LbCpf1 wild type protein sequence is as follows:

LbCpf1-Type V CRISPR-associated protein Cpf1 [Lachnospiraceae bacterium ND2006], GenBank Acc No. WP 051666128.1 (SEQ ID NO: 1)    1

  61 RAEDYKGVKK LLDRYYLSFI NDVLHSIKLK NLNNYISLER KKTRTEKENK ELENLEINLR  121 KEIAKAFKGN EGYKSLFKKD IIETILPEFL DDKDEIALVN SENGFTTAFT GFFDNRENME  181 SEEAKSTSIA FRCINENLTR YISNMDIFEK VDAIFDKHEV QEIKEKILNS DYDVEDFFEG  241 EFFNFVLTQE GIDVYNAIIG GFVTESGEKI KGLNEYINLY NQKTKQKLPK FKPLYKQVLS  301 DRESLSFYGE GYTSDEEVLE VERNTLNKNS EIFSSIKKLE KLFKNFDEYS SAGIFVKNGP  361 AISTISKDIF GEWNVIRDKW NAEYDDIHLK KKAVVTEKYE DDRRKSFKKI GSFSLEQLQE  421 YADADLSVVE KLKEIIIQKV DEIYKVYGSS EKLFDADFVL EKSLKKNDAV VAIMKDLLDS  481 VKSFENYIKA FFGEGKETNR DESFYGDEVL AYDILLKVDH IYDAIRNYVT QKPYSKDKFK  541 LYFQNPQFMG GWDKDKETDY RATILRYGSK YYLAIMDKKY AKCLQKIDKD DVNGNYEKIN  601 YKLLPGPNKM LPKVFFSKKW MAYYNPSEDI QKIYKNGTEK KGDMENLNDC HKLIDFFKDS  661 ISRYPKWSNA YDENESETEK YKDIAGFYRE VEEQGYKVSF ESASKKEVDK LVEEGKLYMF  721 QIYNKDESDK SHGTPNLHTM YFKLLEDENN HGQIRLSGGA ELFMRRASLK KEELVVHPAN  781 SPIANKNPDN PKKTTTLSYD VYKDKRFSED QYELHIPIAI NKCPKNIFKI NTEVRVLLKH  841 DDNPYVIGID RGERNLLYIV VVDGKGNIVE QYSLNEIINN FNGIRIKTDY HSLLDKKEKE  901 RFEARQNWTS IENIKELKAG YISQVVHKIC ELVEKYDAVI ALEDLNSGFK NSRVKVEKQV  961 YQKFEKMLID KLNYMVDKKS NPCATGGALK GYQITNKFES FKSMSTQNGF IFYIPAWLTS 1021 KIDPSTGFVN LLKTKYTSIA DSKKFISSED RIMYVPEEDL FEFALDYKNF SRTDADYIKK 1081 WKLYSYGNRI RIFRNPKKNN VEDWEEVCLT SAYKELENKY GINYQQGDIR ALLCEQSDKA 1141 FYSSFMALMS LMLQMRNSIT GRTDVDFLIS PVKNSDGIFY DSRNYEAQEN AILPKNADAN 1201 GAYNIARKVL WAIGQFKKAE DEKLDKVKIA ISNKEWLEYA QTSVKH  Mature LbCpf1 without 18 amino acid signal sequence: (SEQ ID NO: 2) MSKLEKFTNCYSLSKTLRFKAIPVGKTQENIDNKRLLVEDEKRAEDYKGVKKLL DRYYLSFINDVLHSIKLKNLNNYISLFRKKTRTEKENKELENLEINLRKEIAKAFK GNEGYKSLFKKDIIETILPEFLDDKDEIALVNSFNGFTTAFTGFFDNRENMFSEEA KSTSIAFRCINENLTRYISNMDIFEKVDAIFDKHEVQEIKEKILNSDYDVEDFFEGE FFNFVLTQEGIDVYNAIIGGFVTESGEKIKGLNEYINLYNQKTKQKLPKFKPLYKQ VLSDRESLSFYGEGYTSDEEVLEVFRNTLNKNSEIFSSIKKLEKLFKNFDEYSSAG IFVKNGPAISTISKDIFGEWNVIRDKWNAEYDDIHLKKKAVVTEKYEDDRRKSFK KIGSFSLEQLQEYADADLSVVEKLKEIIIQKVDEIYKVYGSSEKLFDADFVLEKSL KKNDAVVAIMKDLLDSVKSFENYIKAFFGEGKETNRDESFYGDFVLAYDILLKV DHIYDAIRNYVTQKPYSKDKFKLYFQNPQFMGGWDKDKETDYRATILRYGSKY YLAIMDKKYAKCLQKIDKDDVNGNYEKINYKLLPGPNKMLPKVFFSKKWMAY YNPSEDIQKIYKNGTFKKGDMFNLNDCHKLIDFFKDSISRYPKWSNAYDFNFSET EKYKDIAGFYREVEEQGYKVSFESASKKEVDKLVEEGKLYMFQIYNKDFSDKSH GTPNLHTMYFKLLFDENNHGQIRLSGGAELFMRRASLKKEELVVHPANSPIANK NPDNPKKTTTLSYDVYKDKRFSEDQYELHIPIAINKCPKNIFKINTEVRVLLKHD DNPYVIGIDRGERNLLYIVVVDGKGNIVEQYSLNEIINNFNGIRIKTDYHSLLDKK EKERFEARQNWTSIENIKELKAGYISQVVHKICELVEKYDAVIALEDLNSGFKNS RVKVEKQVYQKFEKMLIDKLNYMVDKKSNPCATGGALKGYQITNKFESFKSMS TQNGFIFYIPAWLTSKIDPSTGFVNLLKTKYTSIADSKKFISSFDRIMYVPEEDLFEF ALDYKNFSRTDADYIKKWKLYSYGNRIRIFRNPKKNNVFDWEEVCLTSAYKELF NKYGINYQQGDIRALLCEQSDKAFYSSFMALMSLMLQMRNSITGRTDVDFLISP VKNSDGIFYDSRNYEAQENAILPKNADANGAYNIARKVLWAIGQFKKAEDEKL DKVKIAISNKEWLEYAQTSVKH 

The LbCpf1 variants described herein can include the amino acid sequence of SEQ ID NO:1, e.g., at least comprising amino acids 23-1246 of SEQ ID NO:1, with mutations (i.e., replacement of the native amino acid with a different amino acid, e.g., alanine, glycine, or serine), at one or more of the positions in Table A; amino acids 19-1246 of SEQ ID NO:1 are identical to amino acids 1-1228 of SEQ ID NO:2 (amino acids 1-1246 of SEQ ID NO:1 are referred to herein as LbCPF1 (+18)). In some embodiments, the LbCpf1 variants are at least 80%, e.g., at least 85%, 90%, or 95% identical to the amino acid sequence of SEQ ID NO:2, e.g., have differences at up to 5%, 10%, 15%, or 20% of the residues of SEQ ID NO:2 replaced, e.g., with conservative mutations, in addition to the mutations described herein. In preferred embodiments, the variant retains desired activity of the parent, e.g., the nuclease activity (except where the parent is a nickase or a dead Cpf1), and/or the ability to interact with a guide RNA and target DNA). The version of LbCpf1 used in the present working examples is SEQ ID NO:2, omitting the first 18 amino acids boxed above as described in Zetsche et al. Cell 163, 759-771 (2015).

In some embodiments, the Cpf1 variants also include one of the following mutations listed in Table A, which reduce or destroy the nuclease activity of the Cpf1 (i.e., render them catalytically inactive):

TABLE A Residues involved in DNA and RNA catalysis LbCpf1 (+18) LbCpf1 DNA targeting D850 D832 E853 E835 N855 N837 Y858 Y840 E943 E925 R1156 R1138 S1158 S1140 D1166 D1148 D1198 D1180 RNA processing H777 H759 K786 K768 K803 K785 F807 F789 Mutations that turn Cpf1 into a nickase R1156A R1138A See, e.g., Yamano et al., Cell. 2016 May 5; 165(4):949-62; Fonfara et al., Nature. 2016 Apr. 28; 532(7600):517-21; Dong et al., Nature. 2016 Apr. 28; 532(7600):522-6; and Zetsche et al., Cell. 2015 Oct. 22; 163(3):759-71. Note that “LbCpf1 (+18)” refers to the full sequence of amino acids 1-1246 of SEQ ID NO:1, while the LbCpf1 refers to the sequence of LbCpf1 in Zetsche et al., also shown herein as amino acids 1-1228 of SEQ ID NO:2 and amino acids 19-1246 of SEQ ID NO:1. Thus, in some embodiments, for LbCpf1 catalytic activity-destroying mutations are made at D832 and E925, e.g., D832A and E925A.

The Cpf1 variants, preferably comprising one or more nuclease-reducing or killing mutation, can be fused on the N or C terminus of the Cpf1 to a transcriptional activation domain (e.g., a transcriptional activation domain from the VP16 domain form herpes simplex virus (Sadowski et al., 1988, Nature, 335:563-564) or VP64; the p65 domain from the cellular transcription factor NF-kappaB (Ruben et al., 1991, Science, 251:1490-93); a tripartite effector fused to dCas9, composed of activators VP64, p65, and Rta (VPR) linked in tandem, Chavez et al., Nat Methods. 2015 April; 12(4):326-8); or the p300 HAT domain. p300/CBP is a histone acetyltransferase (HAT) whose function is critical for regulating gene expression in mammalian cells. The p300 HAT domain (1284-1673) is catalytically active and can be fused to nucleases for targeted epigenome editing. See Hilton et al., Nat Biotechnol. 2015 May; 33(5):510-7.

Any inducible protein dimerizing system can be used, e.g., based on the FK506-binding protein (FKBP), see, e.g., Rollins et al., Proc Natl Acad Sci USA. 2000 Jun. 20; 97(13): 7096-7101; the iDIMERIZE™ Inducible Heterodimer System from Clontech/Takara, wherein the proteins of interest are fused to the DmrA and DmrC binding domains respectively, and dimerization is induced by adding the A/C Heterodimerizer (AP21967). Others are also known, e.g., FKBP with CyP-Fas and FKCsA dimerizing agent (see Belshaw et al., Proceedings of the National Academy of Sciences of the United States of America. 93 (10): 4604-7 (1996)); FKBP and FRB domain of mTOR with Rapamycin dimerizing agent (Rivera et al., Nature Medicine. 2 (9): 1028-32 (1996)); GyrB domain with coumermycin dimerizing agent (Farrar et al., Nature. 383 (6596): 178-81 (1996)); gibberellin-induced dimerization (see Miyamoto et al., Nature Chemical Biology. 8 (5): 465-70 (2012); Miyamoto et al., Nature Chemical Biology. 8 (5): 465-70 (2012)); and protein heterodimerization system based on small molecules cross-linking fusion proteins derived from HaloTags and SNAP-tags (Erhart et al., Chemistry and Biology. 20 (4): 549-57 (2013).

To determine the percent identity of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). The length of a reference sequence aligned for comparison purposes is at least 80% of the length of the reference sequence, and in some embodiments is at least 90% or 100%. The nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein nucleic acid “identity” is equivalent to nucleic acid “homology”). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. Percent identity between two polypeptides or nucleic acid sequences is determined in various ways that are within the skill in the art, for instance, using publicly available computer software such as Smith Waterman Alignment (Smith, T. F. and M. S. Waterman (1981) J Mol Biol 147:195-7); “BestFit” (Smith and Waterman, Advances in Applied Mathematics, 482-489 (1981)) as incorporated into GeneMatcher Plusrm, Schwarz and Dayhof (1979) Atlas of Protein Sequence and Structure, Dayhof, M. O., Ed, pp 353-358; BLAST program (Basic Local Alignment Search Tool; (Altschul, S. F., W. Gish, et al. (1990) J Mol Biol 215: 403-10), BLAST-2, BLAST-P, BLAST-N, BLAST-X, WU-BLAST-2, ALIGN, ALIGN-2, CLUSTAL, or Megalign (DNASTAR) software. In addition, those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the length of the sequences being compared. In general, for proteins or nucleic acids, the length of comparison can be any length, up to and including full length (e.g., 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%). For purposes of the present compositions and methods, at least 80% of the full length of the sequence is aligned.

For purposes of the present invention, the comparison of sequences and determination of percent identity between two sequences can be accomplished using a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.

In some embodiments, the mutants have alanine in place of the wild type amino acid. In some embodiments, the mutants have any amino acid other than arginine or lysine (or the native amino acid).

Also provided herein are isolated nucleic acids encoding the Cpf1 fusion proteins, vectors comprising the isolated nucleic acids, optionally operably linked to one or more regulatory domains for expressing the variant proteins, and host cells, e.g., mammalian host cells, comprising the nucleic acids, and optionally expressing the variant proteins.

The fusion proteins described herein can be used for altering the genome of a cell; the methods generally include expressing the variant proteins in the cells, along with a guide RNA having a region complementary to a selected portion of the genome of the cell. Methods for selectively altering the genome of a cell are known in the art, see, e.g., U.S. Pat. No. 8,993,233; US 20140186958; U.S. Pat. No. 9,023,649; WO/2014/099744; WO 2014/089290; WO2014/144592; WO144288; WO2014/204578; WO2014/152432; WO2115/099850; U.S. Pat. No. 8,697,359; US20160024529; US20160024524; US20160024523; US20160024510; US20160017366; US20160017301; US20150376652; US20150356239; US20150315576; US20150291965; US20150252358; US20150247150; US20150232883; US20150232882; US20150203872; US20150191744; US20150184139; US20150176064; US20150167000; US20150166969; US20150159175; US20150159174; US20150093473; US20150079681; US20150067922; US20150056629; US20150044772; US20150024500; US20150024499; US20150020223; US20140356867; US20140295557; US20140273235; US20140273226; US20140273037; US20140189896; US20140113376; US20140093941; US20130330778; US20130288251; US20120088676; US20110300538; US20110236530; US20110217739; US20110002889; US20100076057; US20110189776; US20110223638; US20130130248; US20150050699; US20150071899; US20150045546; US20150031134; US20150024500; US20140377868; US20140357530; US20140349400; US20140335620; US20140335063; US20140315985; US20140310830; US20140310828; US20140309487; US20140304853; US20140298547; US20140295556; US20140294773; US20140287938; US20140273234; US20140273232; US20140273231; US20140273230; US20140271987; US20140256046; US20140248702; US20140242702; US20140242700; US20140242699; US20140242664; US20140234972; US20140227787; US20140212869; US20140201857; US20140199767; US20140189896; US20140186958; US20140186919; US20140186843; US20140179770; US20140179006; US20140170753; WO/2008/108989; WO/2010/054108; WO/2012/164565; WO/2013/098244; WO/2013/176772; Makarova et al., “Evolution and classification of the CRISPR-Cas systems” 9(6) Nature Reviews Microbiology 467-477 (1-23) (June 2011); Wiedenheft et al., “RNA-guided genetic silencing systems in bacteria and archaea” 482 Nature 331-338 (Feb. 16, 2012); Gasiunas et al., “Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria” 109(39) Proceedings of the National Academy of Sciences USA E2579-E2586 (Sep. 4, 2012); Jinek et al., “A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity” 337 Science 816-821 (Aug. 17, 2012); Carroll, “A CRISPR Approach to Gene Targeting” 20(9) Molecular Therapy 1658-1660 (September 2012); U.S. Appl. No. 61/652,086, filed May 25, 2012; Al-Attar et al., Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs): The Hallmark of an Ingenious Antiviral Defense Mechanism in Prokaryotes, Biol Chem. (2011) vol. 392, Issue 4, pp. 277-289; Hale et al., Essential Features and Rational Design of CRISPR RNAs That Function With the Cas RAMP Module Complex to Cleave RNAs, Molecular Cell, (2012) vol. 45, Issue 3, 292-302.

The fusion proteins described herein can be used in place of or in addition to any of the Cas9 or Cpf1 proteins described in the foregoing references, or in combination with analogous mutations described therein, with a guide RNA appropriate for the selected Cpf1, i.e., with guide RNAs that target selected sequences.

In addition, the fusion proteins described herein can be used in place of the wild-type Cas9, Cpf1 or other Cas9 or Cpf1 mutations (such as the dCpf1 or Cpf1 nickase) as known in the art, e.g., a fusion protein with a heterologous functional domain as described in U.S. Pat. No. 8,993,233; US 20140186958; U.S. Pat. No. 9,023,649; WO/2014/099744; WO 2014/089290; WO2014/144592; WO144288; WO2014/204578; WO2014/152432; WO2115/099850; U.S. Pat. No. 8,697,359; US2010/0076057; US2011/0189776; US2011/0223638; US2013/0130248; WO/2008/108989; WO/2010/054108; WO/2012/164565; WO/2013/098244; WO/2013/176772; US20150050699; US 20150071899 and WO 2014/124284.

In some embodiments, the fusion proteins include a linker between the Cpf1 variant and the heterologous functional domains. Linkers that can be used in these fusion proteins (or between fusion proteins in a concatenated structure) can include any sequence that does not interfere with the function of the fusion proteins. In preferred embodiments, the linkers are short, e.g., 2-20 amino acids, and are typically flexible (i.e., comprising amino acids with a high degree of freedom such as glycine, alanine, and serine). In some embodiments, the linker comprises one or more units consisting of GGGS (SEQ ID NO:3) or GGGGS (SEQ ID NO:4), e.g., two, three, four, or more repeats of the GGGS (SEQ ID NO:3) or GGGGS (SEQ ID NO:4) unit. Other linker sequences can also be used.

In some embodiments, the variant protein includes a cell-penetrating peptide sequence that facilitates delivery to the intracellular space, e.g., HIV-derived TAT peptide, penetratins, transportans, or hCT derived cell-penetrating peptides, see, e.g., Caron et al., (2001) Mol Ther. 3(3):310-8; Langel, Cell-Penetrating Peptides: Processes and Applications (CRC Press, Boca Raton FL 2002); El-Andaloussi et al., (2005) Curr Pharm Des. 11(28):3597-611; and Deshayes et al., (2005) Cell Mol Life Sci. 62(16):1839-49.

Cell penetrating peptides (CPPs) are short peptides that facilitate the movement of a wide range of biomolecules across the cell membrane into the cytoplasm or other organelles, e.g. the mitochondria and the nucleus. Examples of molecules that can be delivered by CPPs include therapeutic drugs, plasmid DNA, oligonucleotides, siRNA, peptide-nucleic acid (PNA), proteins, peptides, nanoparticles, and liposomes. CPPs are generally 30 amino acids or less, are derived from naturally or non-naturally occurring protein or chimeric sequences, and contain either a high relative abundance of positively charged amino acids, e.g. lysine or arginine, or an alternating pattern of polar and non-polar amino acids. CPPs that are commonly used in the art include Tat (Frankel et al., (1988) Cell. 55:1189-1193, Vives et al., (1997) J. Biol. Chem. 272:16010-16017), penetratin (Derossi et al., (1994) J. Biol. Chem. 269:10444-10450), polyarginine peptide sequences (Wender et al., (2000) Proc. Natl. Acad. Sci. USA 97:13003-13008, Futaki et al., (2001) J. Biol. Chem. 276:5836-5840), and transportan (Pooga et al., (1998) Nat. Biotechnol. 16:857-861).

CPPs can be linked with their cargo through covalent or non-covalent strategies. Methods for covalently joining a CPP and its cargo are known in the art, e.g. chemical cross-linking (Stetsenko et al., (2000) J. Org. Chem. 65:4900-4909, Gait et al. (2003) Cell. Mol. Life. Sci. 60:844-853) or cloning a fusion protein (Nagahara et al., (1998) Nat. Med. 4:1449-1453). Non-covalent coupling between the cargo and short amphipathic CPPs comprising polar and non-polar domains is established through electrostatic and hydrophobic interactions.

CPPs have been utilized in the art to deliver potentially therapeutic biomolecules into cells. Examples include cyclosporine linked to polyarginine for immunosuppression (Rothbard et al., (2000) Nature Medicine 6(11):1253-1257), siRNA against cyclin B1 linked to a CPP called MPG for inhibiting tumorigenesis (Crombez et al., (2007) Biochem Soc. Trans. 35:44-46), tumor suppressor p53 peptides linked to CPPs to reduce cancer cell growth (Takenobu et al., (2002) Mol. Cancer Ther. 1(12):1043-1049, Snyder et al., (2004) PLoS Biol. 2:E36), and dominant negative forms of Ras or phosphoinositol 3 kinase (PI3K) fused to Tat to treat asthma (Myou et al., (2003) J. Immunol. 171:4399-4405).

CPPs have been utilized in the art to transport contrast agents into cells for imaging and biosensing applications. For example, green fluorescent protein (GFP) attached to Tat has been used to label cancer cells (Shokolenko et al., (2005) DNA Repair 4(4):511-518). Tat conjugated to quantum dots have been used to successfully cross the blood-brain barrier for visualization of the rat brain (Santra et al., (2005) Chem. Commun. 3144-3146). CPPs have also been combined with magnetic resonance imaging techniques for cell imaging (Liu et al., (2006) Biochem. and Biophys. Res. Comm. 347(1):133-140). See also Ramsey and Flynn, Pharmacol Ther. 2015 Jul. 22. pii: S0163-7258(15)00141-2.

Alternatively or in addition, the variant proteins can include a nuclear localization sequence, e.g., SV40 large T antigen NLS (PKKKRRV (SEQ ID NO:5)) and nucleoplasmin NLS (KRPAATKKAGQAKKKK (SEQ ID NO:6)). Other NLSs are known in the art; see, e.g., Cokol et al., EMBO Rep. 2000 Nov. 15; 1(5): 411-415; Freitas and Cunha, Curr Genomics. 2009 Dec.; 10(8): 550-557.

In some embodiments, the variants include a moiety that has a high affinity for a ligand, for example GST, FLAG or hexahistidine sequences. Such affinity tags can facilitate the purification of recombinant variant proteins.

For methods in which the variant proteins are delivered to cells, the proteins can be produced using any method known in the art, e.g., by in vitro translation, or expression in a suitable host cell from nucleic acid encoding the variant protein; a number of methods are known in the art for producing proteins. For example, the proteins can be produced in and purified from yeast, E. coli, insect cell lines, plants, transgenic animals, or cultured mammalian cells; see, e.g., Palomares et al., “Production of Recombinant Proteins: Challenges and Solutions,” Methods Mol Biol. 2004; 267:15-52. In addition, the variant proteins can be linked to a moiety that facilitates transfer into a cell, e.g., a lipid nanoparticle, optionally with a linker that is cleaved once the protein is inside the cell. See, e.g., LaFountaine et al., Int J Pharm. 2015 Aug. 13; 494(1):180-194.

Expression Systems

To use the Cpf1 fusion proteins described herein, it may be desirable to express them from a nucleic acid that encodes them. This can be performed in a variety of ways. For example, the nucleic acid encoding the Cpf1 fusion proteins can be cloned into an intermediate vector for transformation into prokaryotic or eukaryotic cells for replication and/or expression. Intermediate vectors are typically prokaryote vectors, e.g., plasmids, or shuttle vectors, or insect vectors, for storage or manipulation of the nucleic acid encoding the Cpf1 fusion proteins for production of the Cpf1 fusion proteins. The nucleic acid encoding the Cpf1 fusion proteins can also be cloned into an expression vector, for administration to a plant cell, animal cell, preferably a mammalian cell or a human cell, fungal cell, bacterial cell, or protozoan cell.

To obtain expression, a sequence encoding a Cpf1 fusion protein is typically subcloned into an expression vector that contains a promoter to direct transcription. Suitable bacterial and eukaryotic promoters are well known in the art and described, e.g., in Sambrook et al., Molecular Cloning, A Laboratory Manual (3d ed. 2001); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al., eds., 2010). Bacterial expression systems for expressing the engineered protein are available in, e.g., E. coli, Bacillus sp., and Salmonella (Palva et al., 1983, Gene 22:229-235). Kits for such expression systems are commercially available. Eukaryotic expression systems for mammalian cells, yeast, and insect cells are well known in the art and are also commercially available.

The promoter used to direct expression of a nucleic acid depends on the particular application. For example, a strong constitutive promoter is typically used for expression and purification of fusion proteins. In contrast, when the Cpf1 fusion protein is to be administered in vivo for gene regulation, either a constitutive or an inducible promoter can be used, depending on the particular use of the Cpf1 fusion protein. In addition, a preferred promoter for administration of the Cpf1 fusion protein can be a weak promoter, such as HSV TK or a promoter having similar activity. The promoter can also include elements that are responsive to transactivation, e.g., hypoxia response elements, Gal4 response elements, lac repressor response element, and small molecule control systems such as tetracycline-regulated systems and the RU-486 system (see, e.g., Gossen & Bujard, 1992, Proc. Natl. Acad. Sci. USA, 89:5547; Oligino et al., 1998, Gene Ther., 5:491-496; Wang et al., 1997, Gene Ther., 4:432-441; Neering et al., 1996, Blood, 88:1147-55; and Rendahl et al., 1998, Nat. Biotechnol., 16:757-761).

In addition to the promoter, the expression vector typically contains a transcription unit or expression cassette that contains all the additional elements required for the expression of the nucleic acid in host cells, either prokaryotic or eukaryotic. A typical expression cassette thus contains a promoter operably linked, e.g., to the nucleic acid sequence encoding the Cpf1 fusion proteins, and any signals required, e.g., for efficient polyadenylation of the transcript, transcriptional termination, ribosome binding sites, or translation termination. Additional elements of the cassette may include, e.g., enhancers, and heterologous spliced intronic signals.

The particular expression vector used to transport the genetic information into the cell is selected with regard to the intended use of the Cpf1 fusion proteins, e.g., expression in plants, animals, bacteria, fungus, protozoa, etc. Standard bacterial expression vectors include plasmids such as pBR322 based plasmids, pSKF, pET23D, and commercially available tag-fusion expression systems such as GST and LacZ.

Expression vectors containing regulatory elements from eukaryotic viruses are often used in eukaryotic expression vectors, e.g., SV40 vectors, papilloma virus vectors, and vectors derived from Epstein-Barr virus. Other exemplary eukaryotic vectors include pMSG, pAV009/A+, pMTO10/A+, pMAMneo-5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the SV40 early promoter, SV40 late promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.

The vectors for expressing the Cpf1 fusion proteins can include RNA Pol III promoters to drive expression of the guide RNAs, e.g., the H1, U6 or 7SK promoters. These human promoters allow for expression of Cpf1 fusion proteins in mammalian cells following plasmid transfection.

In some embodiments, a single nucleic acid encoding a plurality of Cpf1 gRNAs is used, e. g., as follows

(SEQ ID NO: 7) GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGA TACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTA AACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAA TAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAA ATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGAT TTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGAA TTTCTACTAAGTGTAGAT[spacer_sequence_1] AATTTCTACTAAGTGTAGAT[spacer_sequence_2] AATTTCTACTAAGTGTAGAT[spacer_sequence_3] AATTTCTACTAAGTGTAGA TTTTTTTT The hU6 promoter is shown in bold above.

The Lb crRNA direct repeats are AATTTCTACTAAGTGTAGAT (SEQ ID NO: 38, shown in italics above. The spacer sequences of 17-20 nts (preferably 20) that direct the Cpf1 to the target gene are indicated as spacer sequence_1, 2, or 3.

Some expression systems have markers for selection of stably transfected cell lines such as thymidine kinase, hygromycin B phosphotransferase, and dihydrofolate reductase. High yield expression systems are also suitable, such as using a baculovirus vector in insect cells, with the gRNA encoding sequence under the direction of the polyhedrin promoter or other strong baculovirus promoters.

The elements that are typically included in expression vectors also include a replicon that functions in E. coli, a gene encoding antibiotic resistance to permit selection of bacteria that harbor recombinant plasmids, and unique restriction sites in nonessential regions of the plasmid to allow insertion of recombinant sequences.

Standard transfection methods are used to produce bacterial, mammalian, yeast or insect cell lines that express large quantities of protein, which are then purified using standard techniques (see, e.g., Colley et al., 1989, J. Biol. Chem., 264:17619-22; Guide to Protein Purification, in Methods in Enzymology, vol. 182 (Deutscher, ed., 1990)). Transformation of eukaryotic and prokaryotic cells are performed according to standard techniques (see, e.g., Morrison, 1977, J. Bacteriol. 132:349-351; Clark-Curtiss & Curtiss, Methods in Enzymology 101:347-362 (Wu et al., eds, 1983).

Any of the known procedures for introducing foreign nucleotide sequences into host cells may be used. These include the use of calcium phosphate transfection, polybrene, protoplast fusion, electroporation, nucleofection, liposomes, microinjection, naked DNA, plasmid vectors, viral vectors, both episomal and integrative, and any of the other well-known methods for introducing cloned genomic DNA, cDNA, synthetic DNA or other foreign genetic material into a host cell (see, e.g., Sambrook et al., supra). It is only necessary that the particular genetic engineering procedure used be capable of successfully introducing at least one gene into the host cell capable of expressing the Cpf1 variant.

The present invention also includes the vectors and cells comprising the vectors, and cells and transgenic animals expressing the fusion proteins.

Examples

The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

Methods

The following materials and methods were used in the Examples below.

Plasmids and oligonucleotides.

A list of plasmids, and related sequences used in this study are found in the Sequences section, below; LbCpf1 crRNA information is in Table 1.

TABLE 1 Single Cpf1 crRNAs Spacer Sequence SEQ Genomic Name with Cpf1 PAM ID NO coordinates HBB_P_1 TTTGTACTGATGG 39 Chr11: 5248505- guide TATGGGGCCAA 5248528 HBB_P_2 TTTGAAGTCCAAC 40 Chr11: 5248452- guide TCCTAAGCCAG 5248475 HBB_P_3 TTTGCAAGTGTAT 41 Chr11: 5248550- guide TTACGTAATAT 5248573 AR_P_1 TTTGAGAGTCTGG 42 ChrX: 66763209- guide ATGAGAAATGC 66763232 AR_P_2 TTTCTACCCTCTT 43 ChrX: 66763260- guide CTCTGCCTTTC 66763283 AR_P_3 TTTGCTCTAGGAA 44 ChrX: 66763299- guide CCCTCAGCCCC 66763322 NPY1R_P_1 TTTCAAGCCTCGG 45 Chr4: 164254005- guide GAAACTGCCCT 164254028 NPY1R_P_2 TTTCTTTGTTTGC 46 Chr4: 164254048- guide AGGTCAGTGCC 164254071 NPY1R_P_3 TTTGGGCTGGCGC 47 Chr4: 164254099- guide TCGAGCTCTCC 164254122

dLbCpf1-p65 and dLbCpf1-VPR plasmids (JG1202 and JG1211, respectively) were constructed by cloning p65 and VPR into dLbCpf1 (MMW1578) using BstZ17I and Not I sites through Gibson assembly. VPR was amplified from SP-dCas9-VPR which was a gift from George Church (Addgene plasmid #63798)⁴⁶. dLbCpf1-DmrA(×1) to dLbCpf1-DmrA(×4) (JG674, JG676, JG693, and YET1000, respectively) were generated by inserting dLbCpf1 into AgeI and XhoI digested constructs that have different numbers of DmrA domains (BPK1019, BPK1033, BPK1140, BPK1179 for dCas9-DmrA(×4) to dCas9-DmrA(×4), respectively) using Gibson cloning method. A previously described plasmid encoding DmrC was digested with NruI and p65 or VPR with G4S-linker were added via Gibson assembly for DmrC-P65 (BPK1169) and DmrC-VPR (MMW948). For constructing single crRNA plasmids, oligonucleotide pairs for crRNA spacers were annealed and ligated into BsmBI-digested LbCpf1 crRNA backbone plasmid, BPK3082 (Addgene #78742)¹⁴. For the cloning of multiplexed crRNAs used in this study, three pairs of oligonucleotides were designed to have overhangs. Each oligonucleotides pair was annealed in the presence of T4 PNK, and all three oligo pairs are ligated to BsmBI and HindIII-digested LbCpf1 crRNA backbone plasmid, BPK3082 in one reaction. Sequences for all oligo pairs are listed in Tables 2A-B.

TABLE 2A Multiplexed Cpf1 crRNAs targeting a single promoter SEQ Oligonucleotides sequences Orien- ID Name Pair # to be ordered tation NO:  HBB Pair1 AGATTACTGATGGTATGGGGCCAAA Top 8 Multi- plexed TAGTAGAAATTTTGGCCCCATACC Bottom 9 ATCAGTA Pair 2 ATTTCTACTAAGTGTAGATAAGTCC Top 10 AACTCCTAAGCCAGAATTTCTACT AA ATCTACACTTAGTAGAAATTCTGGC Bottom 11 TTAGGAGTTGGACTTATCTACACT Pair 3 GTGTAGATCAAGTGTATTTACGTAA Top 12 TATAATTTCTACTAAGTGTAGATTT TTTTTA AGCTTAAAAAAAATCTACACTTAG Bottom 13 TAGAAATTATATTACGTAAATACA CTTG AR_ Pair1 AGATAGAGTCTGGATGAGAAATGCA Top 14 Multi- plexed TAGTAGAAATTGCATTTCTCATCCA Bottom 15 GACTCT Pair 2 ATTTCTACTAAGTGTAGATTACCCT Top 16 CTTCTCTGCCTTTCAATTTCTACTAA ATCTACACTTAGTAGAAATTGAAAG Bottom 17 GCAGAGAAGAGGGTAATCTACACT Pair 3 GTGTAGATCTCTAGGAACCCTCAGC Top 18 CCCAATTTCTACTAAGTGTAGATTT TTTTTA AGCTTAAAAAAAATCTACACTTAGT Bottom 19 AGAAATTGGGGCTGAGGGTTCCTAG AG NPY1R Pair1 AGATAAGCCTCGGGAAACTGCCCTA Top 20 Multi- plexed TAGTAGAAATTAGGGCAGTTTCCC Bottom 21 GAGGCTT Pair 2 ATTTCTACTAAGTGTAGATTTTGTT Top 22 TGCAGGTCAGTGCCAATTTCTACTAA ATCTACACTTAGTAGAAATTGGCAC Bottom 23 TGACCTGCAAACAAAATCTACACT Pair 3 GTGTAGATGGCTGGCGCTCGAGCTC Top 24 TCCAATTTCTACTAAGTGTAGATTT TTTTTA AGCTTAAAAAAAATCTACACTTAG Bottom 25 TAGAAATTGGAGAGCTCGAGCGCCA GCC

TABLE 2B Multiplexed Cpf1 crRNAs targeting multiple promoters Oligonucleotides SEQ sequences to Orien- ID Name Pair # be ordered tation NO:  HBB AR Pair1 AGATTACTGATGGTAT Top 26 GGGGCCAAA NPY1R TAGTAGAAATTTTGGC Bottom 27 Multiplexed CCCATACCATCAGTA Pair 2 ATTTCTACTAAGTGTA Top 28 GATCTCTAGGAACCCT CAGCCCCAATTTCTAC TAA ATCTACACTTAGTAGAA Bottom 29 ATTGGGGCTGAGGGTTC CTAGAGATCTACACT Pair 3 GTGTAGATAAGCCTCGG Top 30 GAAACTGCCCTAATTTC TACTAAGTGTAGATTTT TTTTA AGCTTAAAAAAAATCTA Bottom 31 CACTTAGTAGAAATTAG GGCAGTTTCCCGAGGC TT

Human Cell Culture and Transfection.

HEK293 cells were grown at 37°, in 5% CO2 in Dulbecco's Modified Eagle Medium (DMEM) with 10% fetal bovine serum and 1% penicillin and streptomycin. 750 ng of dLbCpf1-p65/VPR with 250 ng of LbCpf1 crRNAs were co-transfected using a 3 ul of TransIT®-LT1 Transfection Reagent (Mirus, cat #MIR2300) into HEK293 cells in a 12-well plate. 400 ng of dLbCpf1 fused with different numbers of DmrA, 200 ng of DmrC-p65/VPR, and 400 ng of LbCpf1 crRNAs were co-transfected using 3 ul of LT-1 into HEK293 cells in a 12-well plate.

Reverse Transcription Quantitative PCR.

Total RNA was extracted from the transfected cells 72 hours post-transfection using the NucleoSpin® RNA Plus (Clontech, cat #740984.250), and 250 ng of purified RNA was used for cDNA synthesis using High-Capacity RNA-cDNA kit (ThermoFisher, cat #4387406). cDNA was diluted 1:20 and 3 ul of cDNA was used for quantitative PCR (qPCR). qPCR reaction samples were prepared using cDNA, SYBR (ThermoFisher, cat #4385612), and primers detecting each target transcript. Primer sequences are listed in Table 3. qPCR was performed using Roche LightCycler480. When Ct values are over 35, we considered them as 35, because Ct values fluctuate for very low expressed transcripts. Samples that were transfected with LbCpf1 crRNA backbone plasmid, BPK3082 were used as negative controls, and the levels of fold activation over negative controls were normalized to the expression of HPRT1.

TABLE 3 RT-qPCR primers SEQ Primer ID Orien- Target name Sequence NO:  tation Gene oET_173 ATGGTGAGCAGAGTGCCCTATC 32 F NPY1R oET_174 ATGGTCCCTGGCAGTCTCCAAA 33 R oET 175 CCATCGGACTCTCATAGGTTGTC 34 F AR oET_176 GACCTGTACTTATTGTCTCTCATC 35 R oET_225 GCACGTGGATCCTGAGAACT 36 F HBB oET 226 ATTGGACAGCAAGAAAGCGAG 37 R F, Forward; R, Reverse

Example 1. Targeted Human Endogenous Gene Regulation Using Individual crRNAs with dLbCpf1-Based Activators

In initial experiments, we tested whether direct fusions of a catalytically inactive Cpf1 nuclease to transcriptional activation domains (FIG. 1A) could activate endogenous human gene promoters. We targeted the promoters of three different endogenous genes that are not expressed in human HEK293 cells (HBB, AR, and NPY1R) by designing three crRNAs for each promoter (See Tables 2A-B). We used a catalytically inactive nuclease-dead version of Cpf1 from Lachnospiraceae bacterium ND2006 (dLbCpf1) because we and others have shown this has higher nuclease activities compared to the other Cpf1 nuclease reported to be active in human cells (AsCpf1 from Acidaminococcus sp. BV3L6)^(14,36,44). dLbCpf1 fusions to a single human NF-KB p65 activation domain tested with the nine individual crRNAs either failed to increase or only weakly increased transcription from the three human gene target promoters (FIG. 1B), consistent with previously published results with single activation domain fusions to dSpCas9⁴⁶. By contrast, dLbCpf1 fusions to the synthetic multimerized VPR activator (consisting of four copies of the viral-based VP16 activator, the human NF-KB p65 activation domain, and the Epstein-Barr virus R transactivator Rta) could significantly activate transcription with at least one crRNA for each of the three target genes (FIG. 1B). This result is similar to previous experiments with dSpCas9 activators, which demonstrated the need to recruit multiple transcriptional activation domains to achieve efficient upregulation of target gene promoter activity^(46,47).

We also tested a larger series of 32 crRNAs positioned within 1 kb upstream or 500 bp downstream of the TSSs of two additional endogenous genes, CD5 and CD22, which encode cell surface proteins. Most of the 32 crRNAs tested could significantly activate the target gene promoter when positioned between −600 bp upstream and ˜400 bp downstream of the TSSs (FIG. 1C), consistent with results obtained using dSpCas9 activators³. The levels of activation observed with dLbCpf1-based activators are comparable to what might be observed in naturally occurring biological systems and are similar to what has been reported previously for analogous dCas9-based activators⁵.

We next sought to develop chemically inducible, bi-partite dLbCpf1-based transcriptional activators. We envisioned using a dimerization system of fragments of the FK506-binding protein (FKBP) and FKBP-rapamycin-binding protein (FRB) known as the DmrA and DmrC domains, respectively, that interact only in the presence of a rapamycin analog known as the A/C heterodimerizer⁴¹, to split dLbCpf1 activators into two parts that would assemble only in the presence of the A/C drug (FIG. 1D). dLbCpf1 fused to a single DmrA domain together with a DmrC domain fused to either NF-KB p65 or VPR failed to activate transcription of the HBB, AR, or NPY1R genes with any of the single crRNAs that had worked efficiently at these promoters with the direct dLbCpf1 activator fusions (FIG. 1E). Reasoning that increasing the number of DmrA domains linked to dLbCpf1 might increase the efficiency of gene activation, we constructed fusions harboring two, three or four DmrA domains. Testing of these fusions with DmrC-VPR with single crRNAs revealed activation at two of the three endogenous gene promoters (HBB and NPY1R; FIG. 1E), with increasing effects observed with more DmrA domains and with maximum levels reaching approximately half of that observed with direct dLbCpf1-VPR fusions. This maximal level of activation was dependent on the presence of the A/C heterodimerizer drug (FIG. 1E), demonstrating that this system is drug-inducible.

Surprisingly, these dLbCpf1 fusions together with DmrC-p65 could robustly activate transcription from all three target gene promoters using single crRNAs (FIG. 1E), an unexpected finding given the lack of activation observed with direct dLbCpf1-p65 fusions with the same crRNAs (FIG. 1B). For the AR promoter, DmrC-p65 could activate transcription by ˜25-fold compared with the lack of an effect by DmrC-VPR. For the HBB and NPY1R promoters, the levels of activation observed with DmrC-p65 were somewhat less (˜50% and ˜30%, respectively) than those obtained with DmrC-VPR but still robust in absolute terms (˜20-fold and ˜10-fold activation). Maximal activation with DmrC-p65 fusions was again dependent on the presence of A/C heterodimerizer drug. Taken together, these findings demonstrate that p65 can provide an important alternative to VPR for the drug-inducible dLbCpf1 activator platform.

Example 2. Multiplex and Synergistic Regulation of Endogenous Human Genes by dLbCpf1-Based Activators

A major advantage of Cpf1 nuclease relative to Cas9 nuclease is the ability to more easily express more than one guide RNA for multiplex applications. Previous work has shown that multiple crRNAs encoded in a single transcript driven by a U6 promoter can be processed into individual crRNAs by Cpf1 itself (FIG. 2A), enabling multiplex induction of mutagenic genome editing events⁴⁹. We tested whether we could use dLbCpf1-based systems to activate three different endogenous human genes using three crRNAs, each already shown to be active with a dLbCpf1-based activator and all encoded on a single transcript (FIG. 2B). Testing of these multiplex crRNA transcripts revealed that these could be used together with dLbCpf1-VPR direct fusions, with dLbCpf1-DmrA(×4) and DmrC-VPR fusions, and with dLbCpf1-DmrA(×4) and DmrC-p⁶⁵ fusions to mediate transcriptional activation of multiple endogenous gene promoters in human HEK293 cells (FIG. 2C). The magnitudes of activation observed with the multiplex crRNAs were somewhat lower (˜18 to 55%) than those observed with expression of a single crRNA. One possible explanation for this difference might be greater competition for binding to dLbCpf1 fusion proteins due to the presumably three-fold higher levels of crRNA present in cells expressing the multiplex transcript. Nonetheless, these results demonstrate that as many as three crRNAs encoded in a larger multiplex transcript can be used to activate transcription of multiple gene targets using dLbCpf1-based activators.

To extend our findings to another human cell line, we also tested the direct VPR activator fusions and drug-regulated VPR and p65 activators in human U2OS cells, targeting the same genes (HBB, AR, and NPY1R) with MST crRNAs and single crRNAs (FIG. 2D). We observed that HBB and NPY1R were highly upregulated and that HBB activation with DmrC-p65 was even greater than in HEK293s. We did not observe activation of the AR gene but this is likely because its baseline expression is already highly elevated in U2OS cells (with a basal quantitative RT-PCR Ct value of ˜28). Relative differences in the efficacies of crRNAs expressed singly or in MSTs were similar to what we observed in HEK293s.

We also sought to determine whether multiple crRNAs expressed from a single construct are actually active in the same cell. Because our experiments were performed on populations of cells with transient transfection of expression vectors, it is formally possible that the multiplex gene activation we observed above was due to different crRNAs being active in different cells within the population of transfected cells. To rule out this possibility, we reasoned that if multiple crRNAs designed against sites within the same gene promoter are expressed from the same transcript within a single cell, this should lead to synergistic increases in transcription from the target gene promoter (FIG. 2E) (synergistic activation is defined as greater than additive effects or two or more activators acting on the same promoter²).With direct fusions of VPR to dLbCpf1, we observed synergistic activation using three crRNAs expressed in a single transcript for two of the three endogenous gene promoters we examined (HBB and AR; FIG. 2F). For the third gene promoter (NPY1R), we did not observe synergistic activation with the three crRNAs when expressed from the same transcript but did see synergy in a control experiment when they were expressed as separate RNAs introduced simultaneously (FIG. 2F). We also did not observe synergistic activation with direct fusions of p65 to dLbCpf1 at any of the three genes with three crRNAs expressed either from the same transcript or from separate transcripts (FIG. 2F). With the drug-inducible dLbCpf1 and VPR activator system, we again observed synergy at the HBB and AR promoters but only with the transcripts expressed separately at the NPY1R promoter (FIG. 2G). A similar pattern of results was observed with the drug-inducible LbCpf1 and p65 system (FIG. 2G). Importantly, the synergistic activation levels observed with the p65 activation domain on the HBB and AR genes were again higher than comparable experiments performed with the VPR activator (FIG. 2G). Taken together, we conclude that multiple active crRNAs can be expressed from a single transcript in the same cell, albeit with somewhat lower activity than if they are expressed from multiple separate expression vectors.

In addition, we assessed the kinetics of activator effects to the addition and withdrawal of A/C heterodimerizer. We found that maximum activation of the HBB and AR genes was observed ˜25 to 35 hours after the addition of drug (FIG. 2H) and return of activated gene expression to baseline occurred ˜35 to 45 hours after withdrawal of the drug (FIG. 2I). We envision that drug-inducibility could be easily extended to other orthologues and we have successfully used these same strategies to regulate and tune dSpCas9-based activators

Sequences Name Addgene # Description MMW1578 104563 CAG-human dLbCpf1(D832A)-NLS-3xHA BPK1169 CAG-DmrC-NLS-FLAG-P65 MMW948 104565 CAG-DmrC-NLS-FLAG-VPR JG1202 CAG-human dLbCpf1(D832A)-NLS-3xHA-P65 JG1211 104567 CAG-human dLbCpf1(D832A)-NLS-3xHA-VPR JG674 104568 CAG-human dLbCpf1(D832A)-NLS-3xHA-DmrA(X1) JG676 104569 CAG-human dLbCpf1(D832A)-NLS-3xHA-DmrA(X2) JG693 104570 CAG-human dLbCpf1(D832A)-NLS-3xHA-DmrA(X3) YET1000 104571 CAG-human dLbCpf1(D832A)-NLS-3xHA-DmrA(X4) BPK3082 78742 U6-LbCpf1-crRNA-BsmBIcassette

1. MMW1578: CAG-human dLbCpf1(D832A)-NLS-3xHA Human codon optimized dLbCpf1: bold, NLS: italic, 3xHA: lower case (SEQ ID NO: 48) ATGAGCAAGCTGGAGAAGTTTACAAACTGCTACTCCCTGT CTAAGACCCTGAGGTTCAAGGCCATCCCTGTGGGCAAGAC CCAGGAGAACATCGACAATAAGCGGCTGCTGGTGGAGGAC GAGAAGAGAGCCGAGGATTATAAGGGCGTGAAGAAGCTGC TGGATCGCTACTATCTGTCTTTTATCAACGACGTGCTGCA CAGCATCAAGCTGAAGAATCTGAACAATTACATCAGCCTG TTCCGGAAGAAAACCAGAACCGAGAAGGAGAATAAGGAGC TGGAGAACCTGGAGATCAATCTGCGGAAGGAGATCGCCAA GGCCTTCAAGGGCAACGAGGGCTACAAGTCCCTGTTTAAG AAGGATATCATCGAGACAATCCTGCCAGAGTTCCTGGACG ATAAGGACGAGATCGCCCTGGTGAACAGCTTCAATGGCTT TACCACAGCCTTCACCGGCTTCTTTGATAACAGAGAGAAT ATGTTTTCCGAGGAGGCCAAGAGCACATCCATCGCCTTCA GGTGTATCAACGAGAATCTGACCCGCTACATCTCTAATAT GGACATCTTCGAGAAGGTGGACGCCATCTTTGATAAGCAC GAGGTGCAGGAGATCAAGGAGAAGATCCTGAACAGCGACT ATGATGTGGAGGATTTCTTTGAGGGCGAGTTCTTTAACTT TGTGCTGACACAGGAGGGCATCGACGTGTATAACGCCATC ATCGGCGGCTTCGTGACCGAGAGCGGCGAGAAGATCAAGG GCCTGAACGAGTACATCAACCTGTATAATCAGAAAACCAA GCAGAAGCTGCCTAAGTTTAAGCCACTGTATAAGCAGGTG CTGAGCGATCGGGAGTCTCTGAGCTTCTACGGCGAGGGCT ATACATCCGATGAGGAGGTGCTGGAGGTGTTTAGAAACAC CCTGAACAAGAACAGCGAGATCTTCAGCTCCATCAAGAAG CTGGAGAAGCTGTTCAAGAATTTTGACGAGTACTCTAGCG CCGGCATCTTTGTGAAGAACGGCCCCGCCATCAGCACAAT CTCCAAGGATATCTTCGGCGAGTGGAACGTGATCCGGGAC AAGTGGAATGCCGAGTATGACGATATCCACCTGAAGAAGA AGGCCGTGGTGACCGAGAAGTACGAGGACGATCGGAGAAA GTCCTTCAAGAAGATCGGCTCCTTTTCTCTGGAGCAGCTG CAGGAGTACGCCGACGCCGATCTGTCTGTGGTGGAGAAGC TGAAGGAGATCATCATCCAGAAGGTGGATGAGATCTACAA GGTGTATGGCTCCTCTGAGAAGCTGTTCGACGCCGATTTT GTGCTGGAGAAGAGCCTGAAGAAGAACGACGCCGTGGTGG CCATCATGAAGGACCTGCTGGATTCTGTGAAGAGCTTCGA GAATTACATCAAGGCCTTCTTTGGCGAGGGCAAGGAGACA AACAGGGACGAGTCCTTCTATGGCGATTTTGTGCTGGCCT ACGACATCCTGCTGAAGGTGGACCACATCTACGATGCCAT CCGCAATTATGTGACCCAGAAGCCCTACTCTAAGGATAAG TTCAAGCTGTATTTTCAGAACCCTCAGTTCATGGGCGGCT GGGACAAGGATAAGGAGACAGACTATCGGGCCACCATCCT GAGATACGGCTCCAAGTACTATCTGGCCATCATGGATAAG AAGTACGCCAAGTGCCTGCAGAAGATCGACAAGGACGATG TGAACGGCAATTACGAGAAGATCAACTATAAGCTGCTGCC CGGCCCTAATAAGATGCTGCCAAAGGTGTTCTTTTCTAAG AAGTGGATGGCCTACTATAACCCCAGCGAGGACATCCAGA AGATCTACAAGAATGGCACATTCAAGAAGGGCGATATGTT TAACCTGAATGACTGTCACAAGCTGATCGACTTCTTTAAG GATAGCATCTCCCGGTATCCAAAGTGGTCCAATGCCTACG ATTTCAACTTTTCTGAGACAGAGAAGTATAAGGACATCGC CGGCTTTTACAGAGAGGTGGAGGAGCAGGGCTATAAGGTG AGCTTCGAGTCTGCCAGCAAGAAGGAGGTGGATAAGCTGG TGGAGGAGGGCAAGCTGTATATGTTCCAGATCTATAACAA GGACTTTTCCGATAAGTCTCACGGCACACCCAATCTGCAC ACCATGTACTTCAAGCTGCTGTTTGACGAGAACAATCACG GACAGATCAGGCTGAGCGGAGGAGCAGAGCTGTTCATGAG GCGCGCCTCCCTGAAGAAGGAGGAGCTGGTGGTGCACCCA GCCAACTCCCCTATCGCCAACAAGAATCCAGATAATCCCA AGAAAACCACAACCCTGTCCTACGACGTGTATAAGGATAA GAGGTTTTCTGAGGACCAGTACGAGCTGCACATCCCAATC GCCATCAATAAGTGCCCCAAGAACATCTTCAAGATCAATA CAGAGGTGCGCGTGCTGCTGAAGCACGACGATAACCCCTA TGTGATCGGCATCGCCAGGGGCGAGCGCAATCTGCTGTAT ATCGTGGTGGTGGACGGCAAGGGCAACATCGTGGAGCAGT ATTCCCTGAACGAGATCATCAACAACTTCAACGGCATCAG GATCAAGACAGATTACCACTCTCTGCTGGACAAGAAGGAG AAGGAGAGGTTCGAGGCCCGCCAGAACTGGACCTCCATCG AGAATATCAAGGAGCTGAAGGCCGGCTATATCTCTCAGGT GGTGCACAAGATCTGCGAGCTGGTGGAGAAGTACGATGCC GTGATCGCCCTGGAGGACCTGAACTCTGGCTTTAAGAATA GCCGCGTGAAGGTGGAGAAGCAGGTGTATCAGAAGTTCGA GAAGATGCTGATCGATAAGCTGAACTACATGGTGGACAAG AAGTCTAATCCTTGTGCAACAGGCGGCGCCCTGAAGGGCT ATCAGATCACCAATAAGTTCGAGAGCTTTAAGTCCATGTC TACCCAGAACGGCTTCATCTTTTACATCCCTGCCTGGCTG ACATCCAAGATCGATCCATCTACCGGCTTTGTGAACCTGC TGAAAACCAAGTATACCAGCATCGCCGATTCCAAGAAGTT CATCAGCTCCTTTGACAGGATCATGTACGTGCCCGAGGAG GATCTGTTCGAGTTTGCCCTGGACTATAAGAACTTCTCTC GCACAGACGCCGATTACATCAAGAAGTGGAAGCTGTACTC CTACGGCAACCGGATCAGAATCTTCCGGAATCCTAAGAAG AACAACGTGTTCGACTGGGAGGAGGTGTGCCTGACCAGCG CCTATAAGGAGCTGTTCAACAAGTACGGCATCAATTATCA GCAGGGCGATATCAGAGCCCTGCTGTGCGAGCAGTCCGAC AAGGCCTTCTACTCTAGCTTTATGGCCCTGATGAGCCTGA TGCTGCAGATGCGGAACAGCATCACAGGCCGCACCGACGT GGATTTTCTGATCAGCCCTGTGAAGAACTCCGACGGCATC TTCTACGATAGCCGGAACTATGAGGCCCAGGAGAATGCCA TCCTGCCAAAGAACGCCGACGCCAATGGCGCCTATAACAT CGCCAGAAAGGTGCTGTGGGCCATCGGCCAGTTCAAGAAG GCCGAGGACGAGAAGCTGGATAAGGTGAAGATCGCCATCT CTAACAAGGAGTGGCTGGAGTACGCCCAGACCAGCGTGAA GCAC AAAAGGCCGGCGGCCACGAAAAAGGCCGGCCAGGCA AAAAAGAAAAAGGGATCCtacccatacgatgttccagatt acgcttatccctacgacgtgcctgattatgcatacccata tgatgtccccgactatgccTAA 2. BPK1169: CAG-DmrC-NLS-FLAG-P65 DmrC: bold, NLS-Flag: italic, P65: lower case (SEQ ID NO: 50) ATGGGATCCAGAATCCTCTGGCATGAGATGTGGCATGAAG GCCTGGAAGAGGCATCTCGTTTGTACTTTGGGGAAAGGAA CGTGAAAGGCATGTTTGAGGTGCTGGAGCCCTTGCATGCT ATGATGGAACGGGGACCCCAGACTCTGAAGGAAACATCCT TTAATCAGGCCTATGGTCGAGATTTAATGGAGGCCCAAGA GTGGTGCAGGAAGTACATGAAATCAGGGAATGTCAAGGAC CTCCTCCAAGCCTGGGACCTCTATTATCATGTGTTCCGAC GAATCTCAAAGGGCGGCGGATCCCCCAAGAAGAAGAGGAA AGTCTCGAGCGACTACAAAGACCATGACGGTGATTATAAA GATCATGACATCGATTACAAGGATGACGATGACAAGGCTG CAGGAGGCGGTGGAAGCGGGatggagttccagtacctgcc agatacagacgatcgtcaccggattgaggagaaacgtaaa aggacatatgagaccttcaagagcatcatgaagaagagtc ctttcagcggacccaccgacccccggcctccacctcgacg cattgctgtgccttcccgcagctcagcttctgtccccaag ccagcaccccagccctatccctttacgtcatccctgagca ccatcaactatgatgagtttcccaccatggtgtttccttc tgggcagatcagccaggcctcggccttggccccggcccct ccccaagtcctgccccaggctccagcccctgcccctgctc cagccatggtatcagctctggcccaggccccagcccctgt cccagtcctagccccaggccctcctcaggctgtggcccca cctgcccccaagcccacccaggctggggaaggaacgctgt cagaggccctgctgcagctgcagtttgatgatgaagacct gggggccttgcttggcaacagcacagacccagctgtgttc acagacctggcatccgtcgataactccgagtttcagcagc tgctgaaccagggcatacctgtggccccccacacaactga gcccatgctgatggagtaccctgaggctataactcgccta gtgacaggggcccagaggccccccgacccagctcctgctc cactgggggccccggggctccccaatggcctcctttcagg agatgaagacttctcctccattgcggacatggacttctca gccctgctgagtcagatcagctctTAA 3. MMW948: CAG-DmrC-NLS-FLAG-VPR DmrC: bold, NLS-Flag: italic, VPR: lowercase (SEQ ID NO: 49) ATGGGATCCAGA ATCCTCTGGCATGAGATGTGGCATGAAGGCCTGGAAGAGG CATCTCGTTTGTACTTTGGGGAAAGGAACGTGAAAGGCAT GTTTGAGGTGCTGGAGCCCTTGCATGCTATGATGGAACGG GGACCCCAGACTCTGAAGGAAACATCCTTTAATCAGGCCT ATGGTCGAGATTTAATGGAGGCCCAAGAGTGGTGCAGGAA GTACATGAAATCAGGGAATGTCAAGGACCTCCTCCAAGCC TGGGACCTCTATTATCATGTGTTCCGACGAATCTCAAAGG GGGGGGATCCCCCAAGAAGAAGAGGAAAGTCTCGAGCGAC TACAAAGACCATGACGGTGATTATAAAGATCATGACATCG ATTACAAGGATGACGATGACAAGGCTGCAGGAGGCGGTGG AAGCGGGTCGgaggccagcggttccggacgggctgacgca ttggacgattttgatctggatatgctgggaagtgacgccc tcgatgattttgaccttgacatgcttggttcggatgccct tgatgactttgacctcgacatgctcggcagtgacgccctt gatgatttcgacctggacatgctgattaactctagaagtt ccggatctccgaaaaagaaacgcaaagttggtagccagta cctgcccgacaccgacgaccggcaccggatcgaggaaaag cggaagcggacctacgagacattcaagagcatcatgaaga agtcccccttcagcggccccaccgaccctagacctccacc tagaagaatcgccgtgcccagcagatccagcgccagcgtg ccaaaacctgccccccagccttaccccttcaccagcagcc tgagcaccatcaactacgacgagttccctaccatggtgtt ccccagcggccagatctctcaggcctctgctctggctcca gcccctcctcaggtgctgcctcaggctcctgctcctgcac cagctccagccatggtgtctgcactggctcaggcaccagc acccgtgcctgtgctggctcctggacctccacaggctgtg gctccaccagcccctaaacctacacaggccggcgagggca cactgtctgaagctctgctgcagctgcagttcgacgacga ggatctgggagccctgctgggaaacagcaccgatcctgcc gtgttcaccgacctggccagcgtggacaacagcgagttcc agcagctgctgaaccagggcatccctgtggcccctcacac caccgagcccatgctgatggaataccccgaggccatcacc cggctcgtgacaggcgctcagaggcctcctgatccagctc ctgcccctctgggagcaccaggcctgcctaatggactgct gtctggcgacgaggacttcagctctatcgccgatatggat ttctcagccttgctgggctctggcagcggcagccgggatt ccagggaagggatgtttttgccgaagcctgaggccggctc cgctattagtgacgtgtttgagggccgcgaggtgtgccag ccaaaacgaatccggccatttcatcctccaggaagtccat gggccaaccgcccactccccgccagcctcgcaccaacacc aaccggtccagtacatgagccagtcgggtcactgaccccg gcaccagtccctcagccactggatccagcgcccgcagtga ctcccgaggccagtcacctgttggaggatcccgatgaaga gacgagccaggctgtcaaagcccttcgggagatggccgat actgtgattccccagaaggaagaggctgcaatctgtggcc aaatggacctttcccatccgcccccaaggggccatctgga tgagctgacaaccacacttgagtccatgaccgaggatctg aacctggactcacccctgaccccggaattgaacgagattc tggataccttcctgaacgacgagtgcctcttgcatgccat gcatatcagcacaggactgtccatcttcgacacatctctg TTT 4. JG1202: CAG-human dLbCpf1(D832A)-NLS-3xHA-P65 Human codon optimized dLbCpf1: bold, NLS: italic, 3xHA: lower case, P65: lower case and bold (SEQ ID NO: 51) ATGAGCAAGCTGGAGAAGTTTACAAACTGCTACTCCCTGT CTAAGACCCTGAGGTTCAAGGCCATCCCTGTGGGCAAGAC CCAGGAGAACATCGACAATAAGCGGCTGCTGGTGGAGGAC GAGAAGAGAGCCGAGGATTATAAGGGCGTGAAGAAGCTGC TGGATCGCTACTATCTGTCTTTTATCAACGACGTGCTGCA CAGCATCAAGCTGAAGAATCTGAACAATTACATCAGCCTG TTCCGGAAGAAAACCAGAACCGAGAAGGAGAATAAGGAGC TGGAGAACCTGGAGATCAATCTGCGGAAGGAGATCGCCAA GGCCTTCAAGGGCAACGAGGGCTACAAGTCCCTGTTTAAG AAGGATATCATCGAGACAATCCTGCCAGAGTTCCTGGACG ATAAGGACGAGATCGCCCTGGTGAACAGCTTCAATGGCTT TACCACAGCCTTCACCGGCTTCTTTGATAACAGAGAGAAT ATGTTTTCCGAGGAGGCCAAGAGCACATCCATCGCCTTCA GGTGTATCAACGAGAATCTGACCCGCTACATCTCTAATAT GGACATCTTCGAGAAGGTGGACGCCATCTTTGATAAGCAC GAGGTGCAGGAGATCAAGGAGAAGATCCTGAACAGCGACT ATGATGTGGAGGATTTCTTTGAGGGCGAGTTCTTTAACTT TGTGCTGACACAGGAGGGCATCGACGTGTATAACGCCATC ATCGGCGGCTTCGTGACCGAGAGCGGCGAGAAGATCAAGG GCCTGAACGAGTACATCAACCTGTATAATCAGAAAACCAA GCAGAAGCTGCCTAAGTTTAAGCCACTGTATAAGCAGGTG CTGAGCGATCGGGAGTCTCTGAGCTTCTACGGCGAGGGCT ATACATCCGATGAGGAGGTGCTGGAGGTGTTTAGAAACAC CCTGAACAAGAACAGCGAGATCTTCAGCTCCATCAAGAAG CTGGAGAAGCTGTTCAAGAATTTTGACGAGTACTCTAGCG CCGGCATCTTTGTGAAGAACGGCCCCGCCATCAGCACAAT CTCCAAGGATATCTTCGGCGAGTGGAACGTGATCCGGGAC AAGTGGAATGCCGAGTATGACGATATCCACCTGAAGAAGA AGGCCGTGGTGACCGAGAAGTACGAGGACGATCGGAGAAA GTCCTTCAAGAAGATCGGCTCCTTTTCTCTGGAGCAGCTG CAGGAGTACGCCGACGCCGATCTGTCTGTGGTGGAGAAGC TGAAGGAGATCATCATCCAGAAGGTGGATGAGATCTACAA GGTGTATGGCTCCTCTGAGAAGCTGTTCGACGCCGATTTT GTGCTGGAGAAGAGCCTGAAGAAGAACGACGCCGTGGTGG CCATCATGAAGGACCTGCTGGATTCTGTGAAGAGCTTCGA GAATTACATCAAGGCCTTCTTTGGCGAGGGCAAGGAGACA AACAGGGACGAGTCCTTCTATGGCGATTTTGTGCTGGCCT ACGACATCCTGCTGAAGGTGGACCACATCTACGATGCCAT CCGCAATTATGTGACCCAGAAGCCCTACTCTAAGGATAAG TTCAAGCTGTATTTTCAGAACCCTCAGTTCATGGGCGGCT GGGACAAGGATAAGGAGACAGACTATCGGGCCACCATCCT GAGATACGGCTCCAAGTACTATCTGGCCATCATGGATAAG AAGTACGCCAAGTGCCTGCAGAAGATCGACAAGGACGATG TGAACGGCAATTACGAGAAGATCAACTATAAGCTGCTGCC CGGCCCTAATAAGATGCTGCCAAAGGTGTTCTTTTCTAAG AAGTGGATGGCCTACTATAACCCCAGCGAGGACATCCAGA AGATCTACAAGAATGGCACATTCAAGAAGGGCGATATGTT TAACCTGAATGACTGTCACAAGCTGATCGACTTCTTTAAG GATAGCATCTCCCGGTATCCAAAGTGGTCCAATGCCTACG ATTTCAACTTTTCTGAGACAGAGAAGTATAAGGACATCGC CGGCTTTTACAGAGAGGTGGAGGAGCAGGGCTATAAGGTG AGCTTCGAGTCTGCCAGCAAGAAGGAGGTGGATAAGCTGG TGGAGGAGGGCAAGCTGTATATGTTCCAGATCTATAACAA GGACTTTTCCGATAAGTCTCACGGCACACCCAATCTGCAC ACCATGTACTTCAAGCTGCTGTTTGACGAGAACAATCACG GACAGATCAGGCTGAGCGGAGGAGCAGAGCTGTTCATGAG GCGCGCCTCCCTGAAGAAGGAGGAGCTGGTGGTGCACCCA GCCAACTCCCCTATCGCCAACAAGAATCCAGATAATCCCA AGAAAACCACAACCCTGTCCTACGACGTGTATAAGGATAA GAGGTTTTCTGAGGACCAGTACGAGCTGCACATCCCAATC GCCATCAATAAGTGCCCCAAGAACATCTTCAAGATCAATA CAGAGGTGCGCGTGCTGCTGAAGCACGACGATAACCCCTA TGTGATCGGCATCGCCAGGGGCGAGCGCAATCTGCTGTAT ATCGTGGTGGTGGACGGCAAGGGCAACATCGTGGAGCAGT ATTCCCTGAACGAGATCATCAACAACTTCAACGGCATCAG GATCAAGACAGATTACCACTCTCTGCTGGACAAGAAGGAG AAGGAGAGGTTCGAGGCCCGCCAGAACTGGACCTCCATCG AGAATATCAAGGAGCTGAAGGCCGGCTATATCTCTCAGGT GGTGCACAAGATCTGCGAGCTGGTGGAGAAGTACGATGCC GTGATCGCCCTGGAGGACCTGAACTCTGGCTTTAAGAATA GCCGCGTGAAGGTGGAGAAGCAGGTGTATCAGAAGTTCGA GAAGATGCTGATCGATAAGCTGAACTACATGGTGGACAAG AAGTCTAATCCTTGTGCAACAGGCGGCGCCCTGAAGGGCT ATCAGATCACCAATAAGTTCGAGAGCTTTAAGTCCATGTC TACCCAGAACGGCTTCATCTTTTACATCCCTGCCTGGCTG ACATCCAAGATCGATCCATCTACCGGCTTTGTGAACCTGC TGAAAACCAAGTATACCAGCATCGCCGATTCCAAGAAGTT CATCAGCTCCTTTGACAGGATCATGTACGTGCCCGAGGAG GATCTGTTCGAGTTTGCCCTGGACTATAAGAACTTCTCTC GCACAGACGCCGATTACATCAAGAAGTGGAAGCTGTACTC CTACGGCAACCGGATCAGAATCTTCCGGAATCCTAAGAAG AACAACGTGTTCGACTGGGAGGAGGTGTGCCTGACCAGCG CCTATAAGGAGCTGTTCAACAAGTACGGCATCAATTATCA GCAGGGCGATATCAGAGCCCTGCTGTGCGAGCAGTCCGAC AAGGCCTTCTACTCTAGCTTTATGGCCCTGATGAGCCTGA TGCTGCAGATGCGGAACAGCATCACAGGCCGCACCGACGT GGATTTTCTGATCAGCCCTGTGAAGAACTCCGACGGCATC TTCTACGATAGCCGGAACTATGAGGCCCAGGAGAATGCCA TCCTGCCAAAGAACGCCGACGCCAATGGCGCCTATAACAT CGCCAGAAAGGTGCTGTGGGCCATCGGCCAGTTCAAGAAG GCCGAGGACGAGAAGCTGGATAAGGTGAAGATCGCCATCT CTAACAAGGAGTGGCTGGAGTACGCCCAGACCAGCGTGAA GCAC AAAAGGCCGGCGGCCACGAAAAAGGCCGGCCAGGCA AAAAAGAAAAAGGGATCC tacccatacgatgttccagatt acgcttatccctacgacgtgcctgattatgcatacccata tgatgtccccgactatgccGGAAGCatggagttccagtac ctgccagatacagacgatcgtcaccggattgaggagaaac gtaaaaggacatatgagaccttcaagagcatcatgaagaa gagtcctttcagcggacccaccgacccccggcctccacct cgacgcattgctgtgccttcccgcagctcagcttctgtcc ccaagccagcaccccagccctatccctttacgtcatccct gagcaccatcaactatgatgagtttcccaccatggtgttt ccttctgggcagatcagccaggcctcggccttggccccgg cccctccccaagtcctgccccaggctccagcccctgcccc tgctccagccatggtatcagctctggcccaggccccagcc cctgtcccagtcctagccccaggccctcctcaggctgtgg ccccacctgcccccaagcccacccaggctggggaaggaac gctgtcagaggccctgctgcagctgcagtttgatgatgaa gacctgggggccttgcttggcaacagcacagacccagctg tgttcacagacctggcatccgtcgataactccgagtttca gcagctgctgaaccagggcatacctgtggccccccacaca actgagcccatgctgatggagtaccctgaggctataactc gcctagtgacaggggcccagaggccccccgacccagctcc tgctccactgggggccccggggctccccaatggcctcctt tcaggagatgaagacttctcctccattgcggacatggact tctcagccctgctgagtcagatcagctctTAA 5. JG1211: CAG-human dLbCpf1(D832A)-NLS-3xHA-VPR Human codon optimized dLbCpf1: bold, NLS: italic, 3xHA: lower case, VPR: lower case and bold (SEQ ID NO: 52) ATGAGCAAGCTGGAGAAGTTTACAAACTGCTACTCCCTGT CTAAGACCCTGAGGTTCAAGGCCATCCCTGTGGGCAAGAC CCAGGAGAACATCGACAATAAGCGGCTGCTGGTGGAGGAC GAGAAGAGAGCCGAGGATTATAAGGGCGTGAAGAAGCTGC TGGATCGCTACTATCTGTCTTTTATCAACGACGTGCTGCA CAGCATCAAGCTGAAGAATCTGAACAATTACATCAGCCTG TTCCGGAAGAAAACCAGAACCGAGAAGGAGAATAAGGAGC TGGAGAACCTGGAGATCAATCTGCGGAAGGAGATCGCCAA GGCCTTCAAGGGCAACGAGGGCTACAAGTCCCTGTTTAAG AAGGATATCATCGAGACAATCCTGCCAGAGTTCCTGGACG ATAAGGACGAGATCGCCCTGGTGAACAGCTTCAATGGCTT TACCACAGCCTTCACCGGCTTCTTTGATAACAGAGAGAAT ATGTTTTCCGAGGAGGCCAAGAGCACATCCATCGCCTTCA GGTGTATCAACGAGAATCTGACCCGCTACATCTCTAATAT GGACATCTTCGAGAAGGTGGACGCCATCTTTGATAAGCAC GAGGTGCAGGAGATCAAGGAGAAGATCCTGAACAGCGACT ATGATGTGGAGGATTTCTTTGAGGGCGAGTTCTTTAACTT TGTGCTGACACAGGAGGGCATCGACGTGTATAACGCCATC ATCGGCGGCTTCGTGACCGAGAGCGGCGAGAAGATCAAGG GCCTGAACGAGTACATCAACCTGTATAATCAGAAAACCAA GCAGAAGCTGCCTAAGTTTAAGCCACTGTATAAGCAGGTG CTGAGCGATCGGGAGTCTCTGAGCTTCTACGGCGAGGGCT ATACATCCGATGAGGAGGTGCTGGAGGTGTTTAGAAACAC CCTGAACAAGAACAGCGAGATCTTCAGCTCCATCAAGAAG CTGGAGAAGCTGTTCAAGAATTTTGACGAGTACTCTAGCG CCGGCATCTTTGTGAAGAACGGCCCCGCCATCAGCACAAT CTCCAAGGATATCTTCGGCGAGTGGAACGTGATCCGGGAC AAGTGGAATGCCGAGTATGACGATATCCACCTGAAGAAGA AGGCCGTGGTGACCGAGAAGTACGAGGACGATCGGAGAAA GTCCTTCAAGAAGATCGGCTCCTTTTCTCTGGAGCAGCTG CAGGAGTACGCCGACGCCGATCTGTCTGTGGTGGAGAAGC TGAAGGAGATCATCATCCAGAAGGTGGATGAGATCTACAA GGTGTATGGCTCCTCTGAGAAGCTGTTCGACGCCGATTTT GTGCTGGAGAAGAGCCTGAAGAAGAACGACGCCGTGGTGG CCATCATGAAGGACCTGCTGGATTCTGTGAAGAGCTTCGA GAATTACATCAAGGCCTTCTTTGGCGAGGGCAAGGAGACA AACAGGGACGAGTCCTTCTATGGCGATTTTGTGCTGGCCT ACGACATCCTGCTGAAGGTGGACCACATCTACGATGCCAT CCGCAATTATGTGACCCAGAAGCCCTACTCTAAGGATAAG TTCAAGCTGTATTTTCAGAACCCTCAGTTCATGGGCGGCT GGGACAAGGATAAGGAGACAGACTATCGGGCCACCATCCT GAGATACGGCTCCAAGTACTATCTGGCCATCATGGATAAG AAGTACGCCAAGTGCCTGCAGAAGATCGACAAGGACGATG TGAACGGCAATTACGAGAAGATCAACTATAAGCTGCTGCC CGGCCCTAATAAGATGCTGCCAAAGGTGTTCTTTTCTAAG AAGTGGATGGCCTACTATAACCCCAGCGAGGACATCCAGA AGATCTACAAGAATGGCACATTCAAGAAGGGCGATATGTT TAACCTGAATGACTGTCACAAGCTGATCGACTTCTTTAAG GATAGCATCTCCCGGTATCCAAAGTGGTCCAATGCCTACG ATTTCAACTTTTCTGAGACAGAGAAGTATAAGGACATCGC CGGCTTTTACAGAGAGGTGGAGGAGCAGGGCTATAAGGTG AGCTTCGAGTCTGCCAGCAAGAAGGAGGTGGATAAGCTGG TGGAGGAGGGCAAGCTGTATATGTTCCAGATCTATAACAA GGACTTTTCCGATAAGTCTCACGGCACACCCAATCTGCAC ACCATGTACTTCAAGCTGCTGTTTGACGAGAACAATCACG GACAGATCAGGCTGAGCGGAGGAGCAGAGCTGTTCATGAG GCGCGCCTCCCTGAAGAAGGAGGAGCTGGTGGTGCACCCA GCCAACTCCCCTATCGCCAACAAGAATCCAGATAATCCCA AGAAAACCACAACCCTGTCCTACGACGTGTATAAGGATAA GAGGTTTTCTGAGGACCAGTACGAGCTGCACATCCCAATC GCCATCAATAAGTGCCCCAAGAACATCTTCAAGATCAATA CAGAGGTGCGCGTGCTGCTGAAGCACGACGATAACCCCTA TGTGATCGGCATCGCCAGGGGCGAGCGCAATCTGCTGTAT ATCGTGGTGGTGGACGGCAAGGGCAACATCGTGGAGCAGT ATTCCCTGAACGAGATCATCAACAACTTCAACGGCATCAG GATCAAGACAGATTACCACTCTCTGCTGGACAAGAAGGAG AAGGAGAGGTTCGAGGCCCGCCAGAACTGGACCTCCATCG AGAATATCAAGGAGCTGAAGGCCGGCTATATCTCTCAGGT GGTGCACAAGATCTGCGAGCTGGTGGAGAAGTACGATGCC GTGATCGCCCTGGAGGACCTGAACTCTGGCTTTAAGAATA GCCGCGTGAAGGTGGAGAAGCAGGTGTATCAGAAGTTCGA GAAGATGCTGATCGATAAGCTGAACTACATGGTGGACAAG AAGTCTAATCCTTGTGCAACAGGCGGCGCCCTGAAGGGCT ATCAGATCACCAATAAGTTCGAGAGCTTTAAGTCCATGTC TACCCAGAACGGCTTCATCTTTTACATCCCTGCCTGGCTG ACATCCAAGATCGATCCATCTACCGGCTTTGTGAACCTGC TGAAAACCAAGTATACCAGCATCGCCGATTCCAAGAAGTT CATCAGCTCCTTTGACAGGATCATGTACGTGCCCGAGGAG GATCTGTTCGAGTTTGCCCTGGACTATAAGAACTTCTCTC GCACAGACGCCGATTACATCAAGAAGTGGAAGCTGTACTC CTACGGCAACCGGATCAGAATCTTCCGGAATCCTAAGAAG AACAACGTGTTCGACTGGGAGGAGGTGTGCCTGACCAGCG CCTATAAGGAGCTGTTCAACAAGTACGGCATCAATTATCA GCAGGGCGATATCAGAGCCCTGCTGTGCGAGCAGTCCGAC AAGGCCTTCTACTCTAGCTTTATGGCCCTGATGAGCCTGA TGCTGCAGATGCGGAACAGCATCACAGGCCGCACCGACGT GGATTTTCTGATCAGCCCTGTGAAGAACTCCGACGGCATC TTCTACGATAGCCGGAACTATGAGGCCCAGGAGAATGCCA TCCTGCCAAAGAACGCCGACGCCAATGGCGCCTATAACAT CGCCAGAAAGGTGCTGTGGGCCATCGGCCAGTTCAAGAAG GCCGAGGACGAGAAGCTGGATAAGGTGAAGATCGCCATCT CTAACAAGGAGTGGCTGGAGTACGCCCAGACCAGCGTGAA GCAC AAAAGGCCGGCGGCCACGAAAAAGGCCGGCCAGGCA AAAAAGAAAAAGGGATCCtacccatacgatgttccagatt acgcttatccctacgacgtgcctgattatgcatacccata tgatgtccccgactatgccGGAAGCgaggccagcggttcc ggacgggctgacgcattggacgattttgatctggatatgc tgggaagtgacgccctcgatgattttgaccttgacatgct tggttcggatgcccttgatgactttgacctcgacatgctc ggcagtgacgcccttgatgatttcgacctggacatgctga ttaactctagaagttccggatctccgaaaaagaaacgcaa agttggtagccagtacctgcccgacaccgacgaccggcac cggatcgaggaaaagcggaagcggacctacgagacattca agagcatcatgaagaagtcccccttcageggccccaccga ccctagacctccacctagaagaatcgccgtgcccagcaga tccagcgccagcgtgccaaaacctgccccccagccttacc ccttcaccagcagcctgagcaccatcaactacgacgagtt ccctaccatggtgttccccagcggccagatctctcaggcc tctgctctggctccagcccctcctcaggtgctgcctcagg ctcctgctcctgcaccagctccagccatggtgtctgcact ggctcaggcaccagcacccgtgcctgtgctggctcctgga cctccacaggctgtggctccaccagcccctaaacctacac aggccggcgagggcacactgtctgaagctctgctgcagct gcagttcgacgacgaggatctgggagccctgctgggaaac agcaccgatcctgccgtgttcaccgacctggccagcgtgg acaacagcgagttccagcagctgctgaaccagggcatccc tgtggcccctcacaccaccgagcccatgctgatggaatac cccgaggccatcacccggctcgtgacaggcgctcagaggc ctcctgatccagctcctgcccctctgggagcaccaggcct gcctaatggactgctgtctggcgacgaggacttcagctct atcgccgatatggatttctcagccttgctgggctctggca gcggcagccgggattccagggaagggatgtttttgccgaa gcctgaggccggctccgctattagtgacgtgtttgagggc cgcgaggtgtgccagccaaaacgaatccggccatttcatc ctccaggaagtccatgggccaaccgcccactccccgccag cctcgcaccaacaccaaccggtccagtacatgagccagtc gggtcactgaccccggcaccagtccctcagccactggatc cagcgcccgcagtgactcccgaggccagtcacctgttgga ggatcccgatgaagagacgagccaggctgtcaaagccctt cgggagatggccgatactgtgattccccagaaggaagagg ctgcaatctgtggccaaatggacctttcccatccgccccc aaggggccatctggatgagctgacaaccacacttgagtcc atgaccgaggatctgaacctggactcacccctgaccccgg aattgaacgagattctggataccttcctgaacgacgagtg cctcttgcatgccatgcatatcagcacaggactgtccatc ttcgacacatctctgTTT 6. JG674: CAG-human dLbCpf1 (D832A)-NLS-3xHA-DmrA(X1) Human codon optimized dLbCpf1: bold, NLS: italic, 3xHA: lower case, DmrA: lowercase and bold (SEQ ID NO: 53) ATGAGCAAGCTGGAGAAGTTTACAAACTGCTACTCCCTGT CTAAGACCCTGAGGTTCAAGGCCATCCCTGTGGGCAAGAC CCAGGAGAACATCGACAATAAGCGGCTGCTGGTGGAGGAC GAGAAGAGAGCCGAGGATTATAAGGGCGTGAAGAAGCTGC TGGATCGCTACTATCTGTCTTTTATCAACGACGTGCTGCA CAGCATCAAGCTGAAGAATCTGAACAATTACATCAGCCTG TTCCGGAAGAAAACCAGAACCGAGAAGGAGAATAAGGAGC TGGAGAACCTGGAGATCAATCTGCGGAAGGAGATCGCCAA GGCCTTCAAGGGCAACGAGGGCTACAAGTCCCTGTTTAAG AAGGATATCATCGAGACAATCCTGCCAGAGTTCCTGGACG ATAAGGACGAGATCGCCCTGGTGAACAGCTTCAATGGCTT TACCACAGCCTTCACCGGCTTCTTTGATAACAGAGAGAAT ATGTTTTCCGAGGAGGCCAAGAGCACATCCATCGCCTTCA GGTGTATCAACGAGAATCTGACCCGCTACATCTCTAATAT GGACATCTTCGAGAAGGTGGACGCCATCTTTGATAAGCAC GAGGTGCAGGAGATCAAGGAGAAGATCCTGAACAGCGACT ATGATGTGGAGGATTTCTTTGAGGGCGAGTTCTTTAACTT TGTGCTGACACAGGAGGGCATCGACGTGTATAACGCCATC ATCGGCGGCTTCGTGACCGAGAGCGGCGAGAAGATCAAGG GCCTGAACGAGTACATCAACCTGTATAATCAGAAAACCAA GCAGAAGCTGCCTAAGTTTAAGCCACTGTATAAGCAGGTG CTGAGCGATCGGGAGTCTCTGAGCTTCTACGGCGAGGGCT ATACATCCGATGAGGAGGTGCTGGAGGTGTTTAGAAACAC CCTGAACAAGAACAGCGAGATCTTCAGCTCCATCAAGAAG CTGGAGAAGCTGTTCAAGAATTTTGACGAGTACTCTAGCG CCGGCATCTTTGTGAAGAACGGCCCCGCCATCAGCACAAT CTCCAAGGATATCTTCGGCGAGTGGAACGTGATCCGGGAC AAGTGGAATGCCGAGTATGACGATATCCACCTGAAGAAGA AGGCCGTGGTGACCGAGAAGTACGAGGACGATCGGAGAAA GTCCTTCAAGAAGATCGGCTCCTTTTCTCTGGAGCAGCTG CAGGAGTACGCCGACGCCGATCTGTCTGTGGTGGAGAAGC TGAAGGAGATCATCATCCAGAAGGTGGATGAGATCTACAA GGTGTATGGCTCCTCTGAGAAGCTGTTCGACGCCGATTTT GTGCTGGAGAAGAGCCTGAAGAAGAACGACGCCGTGGTGG CCATCATGAAGGACCTGCTGGATTCTGTGAAGAGCTTCGA GAATTACATCAAGGCCTTCTTTGGCGAGGGCAAGGAGACA AACAGGGACGAGTCCTTCTATGGCGATTTTGTGCTGGCCT ACGACATCCTGCTGAAGGTGGACCACATCTACGATGCCAT CCGCAATTATGTGACCCAGAAGCCCTACTCTAAGGATAAG TTCAAGCTGTATTTTCAGAACCCTCAGTTCATGGGCGGCT GGGACAAGGATAAGGAGACAGACTATCGGGCCACCATCCT GAGATACGGCTCCAAGTACTATCTGGCCATCATGGATAAG AAGTACGCCAAGTGCCTGCAGAAGATCGACAAGGACGATG TGAACGGCAATTACGAGAAGATCAACTATAAGCTGCTGCC CGGCCCTAATAAGATGCTGCCAAAGGTGTTCTTTTCTAAG AAGTGGATGGCCTACTATAACCCCAGCGAGGACATCCAGA AGATCTACAAGAATGGCACATTCAAGAAGGGCGATATGTT TAACCTGAATGACTGTCACAAGCTGATCGACTTCTTTAAG GATAGCATCTCCCGGTATCCAAAGTGGTCCAATGCCTACG ATTTCAACTTTTCTGAGACAGAGAAGTATAAGGACATCGC CGGCTTTTACAGAGAGGTGGAGGAGCAGGGCTATAAGGTG AGCTTCGAGTCTGCCAGCAAGAAGGAGGTGGATAAGCTGG TGGAGGAGGGCAAGCTGTATATGTTCCAGATCTATAACAA GGACTTTTCCGATAAGTCTCACGGCACACCCAATCTGCAC ACCATGTACTTCAAGCTGCTGTTTGACGAGAACAATCACG GACAGATCAGGCTGAGCGGAGGAGCAGAGCTGTTCATGAG GCGCGCCTCCCTGAAGAAGGAGGAGCTGGTGGTGCACCCA GCCAACTCCCCTATCGCCAACAAGAATCCAGATAATCCCA AGAAAACCACAACCCTGTCCTACGACGTGTATAAGGATAA GAGGTTTTCTGAGGACCAGTACGAGCTGCACATCCCAATC GCCATCAATAAGTGCCCCAAGAACATCTTCAAGATCAATA CAGAGGTGCGCGTGCTGCTGAAGCACGACGATAACCCCTA TGTGATCGGCATCGCCAGGGGCGAGCGCAATCTGCTGTAT ATCGTGGTGGTGGACGGCAAGGGCAACATCGTGGAGCAGT ATTCCCTGAACGAGATCATCAACAACTTCAACGGCATCAG GATCAAGACAGATTACCACTCTCTGCTGGACAAGAAGGAG AAGGAGAGGTTCGAGGCCCGCCAGAACTGGACCTCCATCG AGAATATCAAGGAGCTGAAGGCCGGCTATATCTCTCAGGT GGTGCACAAGATCTGCGAGCTGGTGGAGAAGTACGATGCC GTGATCGCCCTGGAGGACCTGAACTCTGGCTTTAAGAATA GCCGCGTGAAGGTGGAGAAGCAGGTGTATCAGAAGTTCGA GAAGATGCTGATCGATAAGCTGAACTACATGGTGGACAAG AAGTCTAATCCTTGTGCAACAGGCGGCGCCCTGAAGGGCT ATCAGATCACCAATAAGTTCGAGAGCTTTAAGTCCATGTC TACCCAGAACGGCTTCATCTTTTACATCCCTGCCTGGCTG ACATCCAAGATCGATCCATCTACCGGCTTTGTGAACCTGC TGAAAACCAAGTATACCAGCATCGCCGATTCCAAGAAGTT CATCAGCTCCTTTGACAGGATCATGTACGTGCCCGAGGAG GATCTGTTCGAGTTTGCCCTGGACTATAAGAACTTCTCTC GCACAGACGCCGATTACATCAAGAAGTGGAAGCTGTACTC CTACGGCAACCGGATCAGAATCTTCCGGAATCCTAAGAAG AACAACGTGTTCGACTGGGAGGAGGTGTGCCTGACCAGCG CCTATAAGGAGCTGTTCAACAAGTACGGCATCAATTATCA GCAGGGCGATATCAGAGCCCTGCTGTGCGAGCAGTCCGAC AAGGCCTTCTACTCTAGCTTTATGGCCCTGATGAGCCTGA TGCTGCAGATGCGGAACAGCATCACAGGCCGCACCGACGT GGATTTTCTGATCAGCCCTGTGAAGAACTCCGACGGCATC TTCTACGATAGCCGGAACTATGAGGCCCAGGAGAATGCCA TCCTGCCAAAGAACGCCGACGCCAATGGCGCCTATAACAT CGCCAGAAAGGTGCTGTGGGCCATCGGCCAGTTCAAGAAG GCCGAGGACGAGAAGCTGGATAAGGTGAAGATCGCCATCT CTAACAAGGAGTGGCTGGAGTACGCCCAGACCAGCGTGAA GCAC AAAAGGCCGGCGGCCACGAAAAAGGCCGGCCAGGCA AAAAAGAAAAAGGGATCCtacccatacgatgttccagatt acgcttatccctacgacgtgcctgattatgcatacccata tgatgtccccgactatgccTCGAGCGACTACAAAGACCAT GACGGTGATTATAAAGATCATGACATCGATTACAAGGATG ACGATGACAAGGCTGCAGGAGGCGGTGGAAGCGGGagggg agtgcaggtggaaaccatctccccaggagacgggcgcacc ttccccaagcgcggccagacctgcgtggtgcactacaccg ggatgcttgaagatggaaagaaatttgattcctcccggga cagaaacaagccctttaagtttatgctaggcaagcaggag gtgatccgaggctgggaagaaggggttgcccagatgagtg tgggtcagagagccaaactgactatatctccagattatgc ctatggtgccactgggcacccaggcatcatcccaccacat gccactctcgtcttcgatgtggagcttctaaaactggaaG GATAA 7. JG676: CAG-human dLbCpf1(D832A)- NLS-3xHA-DmrA(X2) Human codon optimized dLbCpf1: bold, NLS: italic, 3xHA: lower case, DmrA: lowercase and bold (SEQ ID NO: 54) ATGAGCAAGCTGGAGAAGTTTACAAACTGCTACTCCCTGT CTAAGACCCTGAGGTTCAAGGCCATCCCTGTGGGCAAGAC CCAGGAGAACATCGACAATAAGCGGCTGCTGGTGGAGGAC GAGAAGAGAGCCGAGGATTATAAGGGCGTGAAGAAGCTGC TGGATCGCTACTATCTGTCTTTTATCAACGACGTGCTGCA CAGCATCAAGCTGAAGAATCTGAACAATTACATCAGCCTG TTCCGGAAGAAAACCAGAACCGAGAAGGAGAATAAGGAGC TGGAGAACCTGGAGATCAATCTGCGGAAGGAGATCGCCAA GGCCTTCAAGGGCAACGAGGGCTACAAGTCCCTGTTTAAG AAGGATATCATCGAGACAATCCTGCCAGAGTTCCTGGACG ATAAGGACGAGATCGCCCTGGTGAACAGCTTCAATGGCTT TACCACAGCCTTCACCGGCTTCTTTGATAACAGAGAGAAT ATGTTTTCCGAGGAGGCCAAGAGCACATCCATCGCCTTCA GGTGTATCAACGAGAATCTGACCCGCTACATCTCTAATAT GGACATCTTCGAGAAGGTGGACGCCATCTTTGATAAGCAC GAGGTGCAGGAGATCAAGGAGAAGATCCTGAACAGCGACT ATGATGTGGAGGATTTCTTTGAGGGCGAGTTCTTTAACTT TGTGCTGACACAGGAGGGCATCGACGTGTATAACGCCATC ATCGGCGGCTTCGTGACCGAGAGCGGCGAGAAGATCAAGG GCCTGAACGAGTACATCAACCTGTATAATCAGAAAACCAA GCAGAAGCTGCCTAAGTTTAAGCCACTGTATAAGCAGGTG CTGAGCGATCGGGAGTCTCTGAGCTTCTACGGCGAGGGCT ATACATCCGATGAGGAGGTGCTGGAGGTGTTTAGAAACAC CCTGAACAAGAACAGCGAGATCTTCAGCTCCATCAAGAAG CTGGAGAAGCTGTTCAAGAATTTTGACGAGTACTCTAGCG CCGGCATCTTTGTGAAGAACGGCCCCGCCATCAGCACAAT CTCCAAGGATATCTTCGGCGAGTGGAACGTGATCCGGGAC AAGTGGAATGCCGAGTATGACGATATCCACCTGAAGAAGA AGGCCGTGGTGACCGAGAAGTACGAGGACGATCGGAGAAA GTCCTTCAAGAAGATCGGCTCCTTTTCTCTGGAGCAGCTG CAGGAGTACGCCGACGCCGATCTGTCTGTGGTGGAGAAGC TGAAGGAGATCATCATCCAGAAGGTGGATGAGATCTACAA GGTGTATGGCTCCTCTGAGAAGCTGTTCGACGCCGATTTT GTGCTGGAGAAGAGCCTGAAGAAGAACGACGCCGTGGTGG CCATCATGAAGGACCTGCTGGATTCTGTGAAGAGCTTCGA GAATTACATCAAGGCCTTCTTTGGCGAGGGCAAGGAGACA AACAGGGACGAGTCCTTCTATGGCGATTTTGTGCTGGCCT ACGACATCCTGCTGAAGGTGGACCACATCTACGATGCCAT CCGCAATTATGTGACCCAGAAGCCCTACTCTAAGGATAAG TTCAAGCTGTATTTTCAGAACCCTCAGTTCATGGGCGGCT GGGACAAGGATAAGGAGACAGACTATCGGGCCACCATCCT GAGATACGGCTCCAAGTACTATCTGGCCATCATGGATAAG AAGTACGCCAAGTGCCTGCAGAAGATCGACAAGGACGATG TGAACGGCAATTACGAGAAGATCAACTATAAGCTGCTGCC CGGCCCTAATAAGATGCTGCCAAAGGTGTTCTTTTCTAAG AAGTGGATGGCCTACTATAACCCCAGCGAGGACATCCAGA AGATCTACAAGAATGGCACATTCAAGAAGGGCGATATGTT TAACCTGAATGACTGTCACAAGCTGATCGACTTCTTTAAG GATAGCATCTCCCGGTATCCAAAGTGGTCCAATGCCTACG ATTTCAACTTTTCTGAGACAGAGAAGTATAAGGACATCGC CGGCTTTTACAGAGAGGTGGAGGAGCAGGGCTATAAGGTG AGCTTCGAGTCTGCCAGCAAGAAGGAGGTGGATAAGCTGG TGGAGGAGGGCAAGCTGTATATGTTCCAGATCTATAACAA GGACTTTTCCGATAAGTCTCACGGCACACCCAATCTGCAC ACCATGTACTTCAAGCTGCTGTTTGACGAGAACAATCACG GACAGATCAGGCTGAGCGGAGGAGCAGAGCTGTTCATGAG GCGCGCCTCCCTGAAGAAGGAGGAGCTGGTGGTGCACCCA GCCAACTCCCCTATCGCCAACAAGAATCCAGATAATCCCA AGAAAACCACAACCCTGTCCTACGACGTGTATAAGGATAA GAGGTTTTCTGAGGACCAGTACGAGCTGCACATCCCAATC GCCATCAATAAGTGCCCCAAGAACATCTTCAAGATCAATA CAGAGGTGCGCGTGCTGCTGAAGCACGACGATAACCCCTA TGTGATCGGCATCGCCAGGGGCGAGCGCAATCTGCTGTAT ATCGTGGTGGTGGACGGCAAGGGCAACATCGTGGAGCAGT ATTCCCTGAACGAGATCATCAACAACTTCAACGGCATCAG GATCAAGACAGATTACCACTCTCTGCTGGACAAGAAGGAG AAGGAGAGGTTCGAGGCCCGCCAGAACTGGACCTCCATCG AGAATATCAAGGAGCTGAAGGCCGGCTATATCTCTCAGGT GGTGCACAAGATCTGCGAGCTGGTGGAGAAGTACGATGCC GTGATCGCCCTGGAGGACCTGAACTCTGGCTTTAAGAATA GCCGCGTGAAGGTGGAGAAGCAGGTGTATCAGAAGTTCGA GAAGATGCTGATCGATAAGCTGAACTACATGGTGGACAAG AAGTCTAATCCTTGTGCAACAGGCGGCGCCCTGAAGGGCT ATCAGATCACCAATAAGTTCGAGAGCTTTAAGTCCATGTC TACCCAGAACGGCTTCATCTTTTACATCCCTGCCTGGCTG ACATCCAAGATCGATCCATCTACCGGCTTTGTGAACCTGC TGAAAACCAAGTATACCAGCATCGCCGATTCCAAGAAGTT CATCAGCTCCTTTGACAGGATCATGTACGTGCCCGAGGAG GATCTGTTCGAGTTTGCCCTGGACTATAAGAACTTCTCTC GCACAGACGCCGATTACATCAAGAAGTGGAAGCTGTACTC CTACGGCAACCGGATCAGAATCTTCCGGAATCCTAAGAAG AACAACGTGTTCGACTGGGAGGAGGTGTGCCTGACCAGCG CCTATAAGGAGCTGTTCAACAAGTACGGCATCAATTATCA GCAGGGCGATATCAGAGCCCTGCTGTGCGAGCAGTCCGAC AAGGCCTTCTACTCTAGCTTTATGGCCCTGATGAGCCTGA TGCTGCAGATGCGGAACAGCATCACAGGCCGCACCGACGT GGATTTTCTGATCAGCCCTGTGAAGAACTCCGACGGCATC TTCTACGATAGCCGGAACTATGAGGCCCAGGAGAATGCCA TCCTGCCAAAGAACGCCGACGCCAATGGCGCCTATAACAT CGCCAGAAAGGTGCTGTGGGCCATCGGCCAGTTCAAGAAG GCCGAGGACGAGAAGCTGGATAAGGTGAAGATCGCCATCT CTAACAAGGAGTGGCTGGAGTACGCCCAGACCAGCGTGAA GCAC AAAAGGCCGGCGGCCACGAAAAAGGCCGGCCAGGCA AAAAAGAAAAAGGGATCCtacccatacgatgttccagatt acgcttatccctacgacgtgcctgattatgcatacccata tgatgtccccgactatgccTCGAGCGACTACAAAGACCAT GACGGTGATTATAAAGATCATGACATCGATTACAAGGATG ACGATGACAAGGCTGCAGGAGGCGGTGGAAGCGGGagggg agtgcaggtggaaaccatctccccaggagacgggcgcacc ttccccaagcgcggccagacctgcgtggtgcactacaccg ggatgcttgaagatggaaagaaatttgattcctcccggga cagaaacaagccctttaagtttatgctaggcaagcaggag gtgatccgaggctgggaagaaggggttgcccagatgagtg tgggtcagagagccaaactgactatatctccagattatgc ctatggtgccactgggcacccaggcatcatcccaccacat gccactctcgtcttcgatgtggagcttctaaaactggaaG GTTCtaggggagtgcaggtggaaaccatctccccaggaga cgggcgcaccttccccaagcgcggccagacctgcgtggtg cactacaccgggatgcttgaagatggaaagaaatttgatt cctcccgggacagaaacaagccctttaagtttatgctagg caagcaggaggtgatccgaggctgggaagaaggggttgcc cagatgagtgtgggtcagagagccaaactgactatatctc cagattatgcctatggtgccactgggcacccaggcatcat cccaccacatgccactctcgtcttcgatgtggagcttcta aaactggaaGGATAA 8. JG693: CAG-human dLbCpf1(D832A)- NLS-3xHA-DmrA(X3) Human codon optimized dLbCpf1: bold, NLS: italic, 3xHA: lower case, DmrA: lowercase and bold (SEQ ID NO: 55) ATGAGCAAGCTGGAGAAGTTTACAAACTGCTACTCCCTGT CTAAGACCCTGAGGTTCAAGGCCATCCCTGTGGGCAAGAC CCAGGAGAACATCGACAATAAGCGGCTGCTGGTGGAGGAC GAGAAGAGAGCCGAGGATTATAAGGGCGTGAAGAAGCTGC TGGATCGCTACTATCTGTCTTTTATCAACGACGTGCTGCA CAGCATCAAGCTGAAGAATCTGAACAATTACATCAGCCTG TTCCGGAAGAAAACCAGAACCGAGAAGGAGAATAAGGAGC TGGAGAACCTGGAGATCAATCTGCGGAAGGAGATCGCCAA GGCCTTCAAGGGCAACGAGGGCTACAAGTCCCTGTTTAAG AAGGATATCATCGAGACAATCCTGCCAGAGTTCCTGGACG ATAAGGACGAGATCGCCCTGGTGAACAGCTTCAATGGCTT TACCACAGCCTTCACCGGCTTCTTTGATAACAGAGAGAAT ATGTTTTCCGAGGAGGCCAAGAGCACATCCATCGCCTTCA GGTGTATCAACGAGAATCTGACCCGCTACATCTCTAATAT GGACATCTTCGAGAAGGTGGACGCCATCTTTGATAAGCAC GAGGTGCAGGAGATCAAGGAGAAGATCCTGAACAGCGACT ATGATGTGGAGGATTTCTTTGAGGGCGAGTTCTTTAACTT TGTGCTGACACAGGAGGGCATCGACGTGTATAACGCCATC ATCGGCGGCTTCGTGACCGAGAGCGGCGAGAAGATCAAGG GCCTGAACGAGTACATCAACCTGTATAATCAGAAAACCAA GCAGAAGCTGCCTAAGTTTAAGCCACTGTATAAGCAGGTG CTGAGCGATCGGGAGTCTCTGAGCTTCTACGGCGAGGGCT ATACATCCGATGAGGAGGTGCTGGAGGTGTTTAGAAACAC CCTGAACAAGAACAGCGAGATCTTCAGCTCCATCAAGAAG CTGGAGAAGCTGTTCAAGAATTTTGACGAGTACTCTAGCG CCGGCATCTTTGTGAAGAACGGCCCCGCCATCAGCACAAT CTCCAAGGATATCTTCGGCGAGTGGAACGTGATCCGGGAC AAGTGGAATGCCGAGTATGACGATATCCACCTGAAGAAGA AGGCCGTGGTGACCGAGAAGTACGAGGACGATCGGAGAAA GTCCTTCAAGAAGATCGGCTCCTTTTCTCTGGAGCAGCTG CAGGAGTACGCCGACGCCGATCTGTCTGTGGTGGAGAAGC TGAAGGAGATCATCATCCAGAAGGTGGATGAGATCTACAA GGTGTATGGCTCCTCTGAGAAGCTGTTCGACGCCGATTTT GTGCTGGAGAAGAGCCTGAAGAAGAACGACGCCGTGGTGG CCATCATGAAGGACCTGCTGGATTCTGTGAAGAGCTTCGA GAATTACATCAAGGCCTTCTTTGGCGAGGGCAAGGAGACA AACAGGGACGAGTCCTTCTATGGCGATTTTGTGCTGGCCT ACGACATCCTGCTGAAGGTGGACCACATCTACGATGCCAT CCGCAATTATGTGACCCAGAAGCCCTACTCTAAGGATAAG TTCAAGCTGTATTTTCAGAACCCTCAGTTCATGGGCGGCT GGGACAAGGATAAGGAGACAGACTATCGGGCCACCATCCT GAGATACGGCTCCAAGTACTATCTGGCCATCATGGATAAG AAGTACGCCAAGTGCCTGCAGAAGATCGACAAGGACGATG TGAACGGCAATTACGAGAAGATCAACTATAAGCTGCTGCC CGGCCCTAATAAGATGCTGCCAAAGGTGTTCTTTTCTAAG AAGTGGATGGCCTACTATAACCCCAGCGAGGACATCCAGA AGATCTACAAGAATGGCACATTCAAGAAGGGCGATATGTT TAACCTGAATGACTGTCACAAGCTGATCGACTTCTTTAAG GATAGCATCTCCCGGTATCCAAAGTGGTCCAATGCCTACG ATTTCAACTTTTCTGAGACAGAGAAGTATAAGGACATCGC CGGCTTTTACAGAGAGGTGGAGGAGCAGGGCTATAAGGTG AGCTTCGAGTCTGCCAGCAAGAAGGAGGTGGATAAGCTGG TGGAGGAGGGCAAGCTGTATATGTTCCAGATCTATAACAA GGACTTTTCCGATAAGTCTCACGGCACACCCAATCTGCAC ACCATGTACTTCAAGCTGCTGTTTGACGAGAACAATCACG GACAGATCAGGCTGAGCGGAGGAGCAGAGCTGTTCATGAG GCGCGCCTCCCTGAAGAAGGAGGAGCTGGTGGTGCACCCA GCCAACTCCCCTATCGCCAACAAGAATCCAGATAATCCCA AGAAAACCACAACCCTGTCCTACGACGTGTATAAGGATAA GAGGTTTTCTGAGGACCAGTACGAGCTGCACATCCCAATC GCCATCAATAAGTGCCCCAAGAACATCTTCAAGATCAATA CAGAGGTGCGCGTGCTGCTGAAGCACGACGATAACCCCTA TGTGATCGGCATCGCCAGGGGCGAGCGCAATCTGCTGTAT ATCGTGGTGGTGGACGGCAAGGGCAACATCGTGGAGCAGT ATTCCCTGAACGAGATCATCAACAACTTCAACGGCATCAG GATCAAGACAGATTACCACTCTCTGCTGGACAAGAAGGAG AAGGAGAGGTTCGAGGCCCGCCAGAACTGGACCTCCATCG AGAATATCAAGGAGCTGAAGGCCGGCTATATCTCTCAGGT GGTGCACAAGATCTGCGAGCTGGTGGAGAAGTACGATGCC GTGATCGCCCTGGAGGACCTGAACTCTGGCTTTAAGAATA GCCGCGTGAAGGTGGAGAAGCAGGTGTATCAGAAGTTCGA GAAGATGCTGATCGATAAGCTGAACTACATGGTGGACAAG AAGTCTAATCCTTGTGCAACAGGCGGCGCCCTGAAGGGCT ATCAGATCACCAATAAGTTCGAGAGCTTTAAGTCCATGTC TACCCAGAACGGCTTCATCTTTTACATCCCTGCCTGGCTG ACATCCAAGATCGATCCATCTACCGGCTTTGTGAACCTGC TGAAAACCAAGTATACCAGCATCGCCGATTCCAAGAAGTT CATCAGCTCCTTTGACAGGATCATGTACGTGCCCGAGGAG GATCTGTTCGAGTTTGCCCTGGACTATAAGAACTTCTCTC GCACAGACGCCGATTACATCAAGAAGTGGAAGCTGTACTC CTACGGCAACCGGATCAGAATCTTCCGGAATCCTAAGAAG AACAACGTGTTCGACTGGGAGGAGGTGTGCCTGACCAGCG CCTATAAGGAGCTGTTCAACAAGTACGGCATCAATTATCA GCAGGGCGATATCAGAGCCCTGCTGTGCGAGCAGTCCGAC AAGGCCTTCTACTCTAGCTTTATGGCCCTGATGAGCCTGA TGCTGCAGATGCGGAACAGCATCACAGGCCGCACCGACGT GGATTTTCTGATCAGCCCTGTGAAGAACTCCGACGGCATC TTCTACGATAGCCGGAACTATGAGGCCCAGGAGAATGCCA TCCTGCCAAAGAACGCCGACGCCAATGGCGCCTATAACAT CGCCAGAAAGGTGCTGTGGGCCATCGGCCAGTTCAAGAAG GCCGAGGACGAGAAGCTGGATAAGGTGAAGATCGCCATCT CTAACAAGGAGTGGCTGGAGTACGCCCAGACCAGCGTGAA GCAC AAAAGGCCGGCGGCCACGAAAAAGGCCGGCCAGGCA AAAAAGAAAAAGGGATCCtacccatacgatgttccagatt acgcttatccctacgacgtgcctgattatgcatacccata tgatgtccccgactatgccTCGAGCGACTACAAAGACCAT GACGGTGATTATAAAGATCATGACATCGATTACAAGGATG ACGATGACAAGGCTGCAGGAGGCGGTGGAAGCGGGagggg agtgcaggtggaaaccatctccccaggagacgggcgcacc ttccccaagcgcggccagacctgcgtggtgcactacaccg ggatgcttgaagatggaaagaaatttgattcctcccggga cagaaacaagccctttaagtttatgctaggcaagcaggag gtgatccgaggctgggaagaaggggttgcccagatgagtg tgggtcagagagccaaactgactatatctccagattatgc ctatggtgccactgggcacccaggcatcatcccaccacat gccactctcgtcttcgatgtggagcttctaaaactggaag gatctggtggaaGCGGGaggggagtgcaggtggaaaccat ctccccaggagacgggcgcaccttccccaagcgcggccag acctgcgtggtgcactacaccgggatgcttgaagatggaa agaaatttgattcctcccgggacagaaacaagccctttaa gtttatgctaggcaagcaggaggtgatccgaggctgggaa gaaggggttgcccagatgagtgtgggtcagagagccaaac tgactatatctccagattatgcctatggtgccactgggca cccaggcatcatcccaccacatgccactctcgtcttcgat gtggagcttctaaaactggaaGGTTCTaggggagtgcagg tggaaaccatctccccaggagacgggcgcaccttccccaa gcgcggccagacctgcgtggtgcactacaccgggatgctt gaagatggaaagaaatttgattcctcccgggacagaaaca agccctttaagtttatgctaggcaagcaggaggtgatccg aggctgggaagaaggggttgcccagatgagtgtgggtcag agagccaaactgactatatctccagattatgcctatggtg ccactgggcacccaggcatcatcccaccacatgccactct cgtcttcgatgtggagcttctaaaactggaaGGATAA 9. YET1000: CAG-human dLbCpf1(D832A)- NLS-3xHA-DmrA(X4) Human codon optimized dLbCpf1: bold, NLS: italic, 3xHA: lower case, DmrA: lowercase and bold (SEQ ID NO: 56) ATGAGCAAGCTGGAGAAGTTTACAAACTGCTACTCCCTGT CTAAGACCCTGAGGTTCAAGGCCATCCCTGTGGGCAAGAC CCAGGAGAACATCGACAATAAGCGGCTGCTGGTGGAGGAC GAGAAGAGAGCCGAGGATTATAAGGGCGTGAAGAAGCTGC TGGATCGCTACTATCTGTCTTTTATCAACGACGTGCTGCA CAGCATCAAGCTGAAGAATCTGAACAATTACATCAGCCTG TTCCGGAAGAAAACCAGAACCGAGAAGGAGAATAAGGAGC TGGAGAACCTGGAGATCAATCTGCGGAAGGAGATCGCCAA GGCCTTCAAGGGCAACGAGGGCTACAAGTCCCTGTTTAAG AAGGATATCATCGAGACAATCCTGCCAGAGTTCCTGGACG ATAAGGACGAGATCGCCCTGGTGAACAGCTTCAATGGCTT TACCACAGCCTTCACCGGCTTCTTTGATAACAGAGAGAAT ATGTTTTCCGAGGAGGCCAAGAGCACATCCATCGCCTTCA GGTGTATCAACGAGAATCTGACCCGCTACATCTCTAATAT GGACATCTTCGAGAAGGTGGACGCCATCTTTGATAAGCAC GAGGTGCAGGAGATCAAGGAGAAGATCCTGAACAGCGACT ATGATGTGGAGGATTTCTTTGAGGGCGAGTTCTTTAACTT TGTGCTGACACAGGAGGGCATCGACGTGTATAACGCCATC ATCGGCGGCTTCGTGACCGAGAGCGGCGAGAAGATCAAGG GCCTGAACGAGTACATCAACCTGTATAATCAGAAAACCAA GCAGAAGCTGCCTAAGTTTAAGCCACTGTATAAGCAGGTG CTGAGCGATCGGGAGTCTCTGAGCTTCTACGGCGAGGGCT ATACATCCGATGAGGAGGTGCTGGAGGTGTTTAGAAACAC CCTGAACAAGAACAGCGAGATCTTCAGCTCCATCAAGAAG CTGGAGAAGCTGTTCAAGAATTTTGACGAGTACTCTAGCG CCGGCATCTTTGTGAAGAACGGCCCCGCCATCAGCACAAT CTCCAAGGATATCTTCGGCGAGTGGAACGTGATCCGGGAC AAGTGGAATGCCGAGTATGACGATATCCACCTGAAGAAGA AGGCCGTGGTGACCGAGAAGTACGAGGACGATCGGAGAAA GTCCTTCAAGAAGATCGGCTCCTTTTCTCTGGAGCAGCTG CAGGAGTACGCCGACGCCGATCTGTCTGTGGTGGAGAAGC TGAAGGAGATCATCATCCAGAAGGTGGATGAGATCTACAA GGTGTATGGCTCCTCTGAGAAGCTGTTCGACGCCGATTTT GTGCTGGAGAAGAGCCTGAAGAAGAACGACGCCGTGGTGG CCATCATGAAGGACCTGCTGGATTCTGTGAAGAGCTTCGA GAATTACATCAAGGCCTTCTTTGGCGAGGGCAAGGAGACA AACAGGGACGAGTCCTTCTATGGCGATTTTGTGCTGGCCT ACGACATCCTGCTGAAGGTGGACCACATCTACGATGCCAT CCGCAATTATGTGACCCAGAAGCCCTACTCTAAGGATAAG TTCAAGCTGTATTTTCAGAACCCTCAGTTCATGGGCGGCT GGGACAAGGATAAGGAGACAGACTATCGGGCCACCATCCT GAGATACGGCTCCAAGTACTATCTGGCCATCATGGATAAG AAGTACGCCAAGTGCCTGCAGAAGATCGACAAGGACGATG TGAACGGCAATTACGAGAAGATCAACTATAAGCTGCTGCC CGGCCCTAATAAGATGCTGCCAAAGGTGTTCTTTTCTAAG AAGTGGATGGCCTACTATAACCCCAGCGAGGACATCCAGA AGATCTACAAGAATGGCACATTCAAGAAGGGCGATATGTT TAACCTGAATGACTGTCACAAGCTGATCGACTTCTTTAAG GATAGCATCTCCCGGTATCCAAAGTGGTCCAATGCCTACG ATTTCAACTTTTCTGAGACAGAGAAGTATAAGGACATCGC CGGCTTTTACAGAGAGGTGGAGGAGCAGGGCTATAAGGTG AGCTTCGAGTCTGCCAGCAAGAAGGAGGTGGATAAGCTGG TGGAGGAGGGCAAGCTGTATATGTTCCAGATCTATAACAA GGACTTTTCCGATAAGTCTCACGGCACACCCAATCTGCAC ACCATGTACTTCAAGCTGCTGTTTGACGAGAACAATCACG GACAGATCAGGCTGAGCGGAGGAGCAGAGCTGTTCATGAG GCGCGCCTCCCTGAAGAAGGAGGAGCTGGTGGTGCACCCA GCCAACTCCCCTATCGCCAACAAGAATCCAGATAATCCCA AGAAAACCACAACCCTGTCCTACGACGTGTATAAGGATAA GAGGTTTTCTGAGGACCAGTACGAGCTGCACATCCCAATC GCCATCAATAAGTGCCCCAAGAACATCTTCAAGATCAATA CAGAGGTGCGCGTGCTGCTGAAGCACGACGATAACCCCTA TGTGATCGGCATCGCCAGGGGCGAGCGCAATCTGCTGTAT ATCGTGGTGGTGGACGGCAAGGGCAACATCGTGGAGCAGT ATTCCCTGAACGAGATCATCAACAACTTCAACGGCATCAG GATCAAGACAGATTACCACTCTCTGCTGGACAAGAAGGAG AAGGAGAGGTTCGAGGCCCGCCAGAACTGGACCTCCATCG AGAATATCAAGGAGCTGAAGGCCGGCTATATCTCTCAGGT GGTGCACAAGATCTGCGAGCTGGTGGAGAAGTACGATGCC GTGATCGCCCTGGAGGACCTGAACTCTGGCTTTAAGAATA GCCGCGTGAAGGTGGAGAAGCAGGTGTATCAGAAGTTCGA GAAGATGCTGATCGATAAGCTGAACTACATGGTGGACAAG AAGTCTAATCCTTGTGCAACAGGCGGCGCCCTGAAGGGCT ATCAGATCACCAATAAGTTCGAGAGCTTTAAGTCCATGTC TACCCAGAACGGCTTCATCTTTTACATCCCTGCCTGGCTG ACATCCAAGATCGATCCATCTACCGGCTTTGTGAACCTGC TGAAAACCAAGTATACCAGCATCGCCGATTCCAAGAAGTT CATCAGCTCCTTTGACAGGATCATGTACGTGCCCGAGGAG GATCTGTTCGAGTTTGCCCTGGACTATAAGAACTTCTCTC GCACAGACGCCGATTACATCAAGAAGTGGAAGCTGTACTC CTACGGCAACCGGATCAGAATCTTCCGGAATCCTAAGAAG AACAACGTGTTCGACTGGGAGGAGGTGTGCCTGACCAGCG CCTATAAGGAGCTGTTCAACAAGTACGGCATCAATTATCA GCAGGGCGATATCAGAGCCCTGCTGTGCGAGCAGTCCGAC AAGGCCTTCTACTCTAGCTTTATGGCCCTGATGAGCCTGA TGCTGCAGATGCGGAACAGCATCACAGGCCGCACCGACGT GGATTTTCTGATCAGCCCTGTGAAGAACTCCGACGGCATC TTCTACGATAGCCGGAACTATGAGGCCCAGGAGAATGCCA TCCTGCCAAAGAACGCCGACGCCAATGGCGCCTATAACAT CGCCAGAAAGGTGCTGTGGGCCATCGGCCAGTTCAAGAAG GCCGAGGACGAGAAGCTGGATAAGGTGAAGATCGCCATCT CTAACAAGGAGTGGCTGGAGTACGCCCAGACCAGCGTGAA GCAC AAAAGGCCGGCGGCCACGAAAAAGGCCGGCCAGGCA AAAAAGAAAAAGGGATCCtacccatacgatgttccagatt acgcttatccctacgacgtgcctgattatgcatacccata tgatgtccccgactatgccTCGAGCGACTACAAAGACCAT GACGGTGATTATAAAGATCATGACATCGATTACAAGGATG ACGATGACAAGGCTGCAGGAGGCGGTGGAAGCGGGGGAag gggagtgcaggtggaaaccatctccccaggagacgggcgc accttccccaagcgcggccagacctgcgtggtgcactaca ccgggatgcttgaagatggaaagaaatttgattcctcccg ggacagaaacaagccctttaagtttatgctaggcaagcag gaggtgatccgaggctgggaagaaggggttgcccagatga gtgtgggtcagagagccaaactgactatatctccagatta tgcctatggtgccactgggcacccaggcatcatcccacca catgccactctcgtcttcgatgtggagcttctaaaactgg aaGGTTCTaggggagtgcaggtggaaaccatctccccagg agacgggcgcaccttccccaagcgcggccagacctgcgtg gtgcactacaccgggatgcttgaagatggaaagaaatttg attcctcccgggacagaaacaagccctttaagtttatgct aggcaagcaggaggtgatccgaggctgggaagaaggggtt gcccagatgagtgtgggtcagagagccaaactgactatat ctccagattatgcctatggtgccactgggcacccaggcat catcccaccacatgccactctcgtcttcgatgtggagctt ctaaaactggaaGGGGGAAGCGGTGGAAGCGGGaggggag tgcaggtggaaaccatctccccaggagacgggcgcacctt ccccaagcgcggccagacctgcgtggtgcactacaccggg atgcttgaagatggaaagaaatttgattcctcccgggaca gaaacaagccctttaagtttatgctaggcaagcaggaggt gatccgaggctgggaagaaggggttgcccagatgagtgtg ggtcagagagccaaactgactatatctccagattatgcct atggtgccactgggcacccaggcatcatcccaccacatgc cactctcgtcttcgatgtggagcttctaaaactggaaGGT TCTaggggagtgcaggtggaaaccatctccccaggagacg ggcgcaccttccccaagcgcggccagacctgcgtggtgca ctacaccgggatgcttgaagatggaaagaaatttgattcc tcccgggacagaaacaagccctttaagtttatgctaggca agcaggaggtgatccgaggctgggaagaaggggttgccca gatgagtgtgggtcagagagccaaactgactatatctcca gattatgcctatggtgccactgggcacccaggcatcatcc caccacatgccactctcgtcttcgatgtggagcttctaaa actggaaGGATAA 10. BPK3082: U6-Lb-crRNA-BsmBlcassette U6 promoter: bold, Lb crRNA: italic, BsmBI sites: lower case, U6 terminator: italic and bold (SEQ ID NO: 57) TGTACAAAAAAGCAGGCTTTAAAGGAACCAATTCAGTCGA CTGGATCCGGTACCAAGGTCGGGCAGGAAGAGGGCCTATT TCCCATGATTCCTTCATATTTGCATATACGATACAAGGCT GTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAA AGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATT TCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATG GACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATT TCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACC GA ATTTCTACTAAGTGTAGATGgagacgATTAATGcgtctcC TTTTTTT 11. FIG. 2b_sequence hU6 promoter: bold, Lb crRNA direct repeats: italic, crRNA1_HBB spacer sequence: lower case, crRNA3_AR spacer sequence: lower case and bold, crRNA1_NPY1R spacer sequence: lower case, bold and italic, terminator: bold and italic (SEQ ID NO: 58) GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATAC GATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGAC TGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGA AAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTAT GTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAA GTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGA CGAAACACCGAATTTCTACTAAGTGTAGATtactgatggt atggggccaaAATTTCTACTAAGTGTAGAT ctctaggaac cctcagcccc AATTTCTACTAAGTGTAGAT aagcctcggg aaactgccct AATTTCTACTAAGTGTAGA TTTTTTTT

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Other Embodiments

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims. 

What is claimed is:
 1. A method of increasing expression of a target gene in a cell, the method comprising contacting the cell with, or expressing in the cell: (a) a first fusion protein comprising a catalytically inactive Lachnospiraceae bacterium ND2006 Cpf1 (dLbCpf1) fused to more than one first dimerization domains, and (b) a second fusion protein comprising at least one activation domain fused to a second dimerization domain that dimerizes with at least one of the first dimerization domains in the first fusion protein in the presence of a dimerizing agent, wherein the first and second dimerization domains are rapamycin analog A/C heterodimerizer DmrA or DmrC, and at least one crRNA that directs the second fusion protein to a regulatory region of the target gene.
 2. The method of claim 1, wherein (i) the first dimerization domain in the first fusion protein is DmrA, and the second dimerization domain in the second fusion protein is DmrC, or (ii) the first dimerization domain in the first fusion protein is DmrC, and the second dimerization domain in the second fusion protein is DmrA.
 3. The method of claim 1, wherein the activation domain is a transcriptional activation domain from herpes simplex viral protein 16 (VP64), Epstein-Barr virus R transactivator (Rta), p65 domain from cellular transcription factor NF-κB (NF-κB p65), or a tripartite effector composed of VP64, p65, and Rta (VPR).
 4. The method of claim 1, further comprising an intervening linker between each of the activation domain(s) or the second dimerization domain.
 5. The method of claim 1, wherein the first fusion protein is fused to two, three or four first dimerization domains.
 6. The method of claim 1, wherein the cell is a mammalian cell.
 7. The method of claim 6, wherein the mammalian cell is a human cell.
 8. The method of claim 1, wherein the regulatory region is a promoter region. 